Sedative effects of oral pregabalin premedication on intravenous sedation using propofol target-controlled infusion

Abstract

Purpose

The sedative effects of pregabalin during perioperative period have not been sufficiently characterized. The aim of this study was to verify the sedative effects of premedication with pregabalin on intravenous sedation (IVS) using propofol and also to assess the influences of this agent on circulation, respiration, and postanesthetic complications.

Methods

Ten healthy young volunteers underwent 1 h of IVS using propofol, three times per subject, on separate days (first time, no pregabalin; second time, pregabalin 100 mg; third time, pregabalin 200 mg). The target blood concentration (C _T) of propofol was increased in a stepwise fashion based on the bispectral index (BIS) value. Ramsay's sedation score (RSS) was determined at each propofol C _T. Propofol C _T was analyzed at each sedation level. Circulation and respiration during IVS and complications were also verified.

Results

Propofol C _T was reduced at BIS values of 60 and 70 in both premedicated groups (100 mg: p = 0.043 and 0.041; 200 mg: p = 0.004 and 0.016, respectively) and at a BIS value of 80 in the pregabalin 200 mg group (p < 0.001). Propofol C _T was decreased at RSS 4–6 in the pregabalin 100 mg group (RSS 4: p = 0.047; RSS 5: p = 0.007; RSS 6: p = 0.014), and at RSS 3–6 in the pregabalin 200 mg group (RSS 3–5: p < 0.001; RSS 6: p = 0.002).

Conclusion

We conclude that oral premedication with pregabalin reduces the amount of propofol required to obtain an acceptable and adequate sedation level.

http://ift.tt/2pX2HIj

Development of a scoring system to predict massive postpartum transfusion in placenta previa totalis

Abstract

Purpose

It is important to predict massive postpartum hemorrhage in patients with placenta previa totalis (PPT) and a method that accurately predicts this event is needed. The present study developed a scoring system that predicts massive transfusion in patients with PPT.

Methods

This single-center retrospective cohort study comprised 238 patients with PPT who underwent caesarean section between January 2004 and December 2010. Massive transfusion was defined as the transfusion of ≥8 units of packed red blood cells within 24 h after delivery. Multivariate regression analysis was used to estimate the risks of massive transfusion. A probability score model was then constructed and tested for performance. Subsequently, the model was validated in other patients with PPT (n = 117).

Results

Thirty-one patients (13.0%) underwent massive transfusion. Ultrasound suspicion of placental adhesion, previous caesarean section, gestational age <37 weeks, sponge-like appearance of the cervix, and anterior placenta were all independent predictors of massive transfusion. The performance for the score model revealed good calibration (Hosmer–Lemeshow chi-squared 1.64; P = 0.44), and its discrimination (the area under the receiver operating characteristic for this model was 0.84) was better than when suspicion of placental adhesion was used alone (0.67; P < 0.001). In the validation set, the performance was 0.88.

Conclusion

The scoring system developed using the five independent risk factors had better performance to predict massive transfusion in patients with PPT than when suspicion of placental adhesion was used alone. However, further large-scale studies are warranted to clarify the usefulness and accuracy of this model.

http://ift.tt/2qCOkFl

MEP monitoring during aortic surgery: what we truly know

http://ift.tt/2qCYLZs

The Greek Version of the Ohkuma Questionnaire for Dysphagia Screening

http://otorhinolaryngology-crete.blogspot.gr/2017/05/the-greek-version-of-ohkuma.html

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com

Adjuvant radiotherapy after radical cystectomy for patients with muscle invasive bladder cancer: a phase II trial

Abstract

Background

Neo-adjuvant chemotherapy followed by radical cystectomy with extended pelvic lymph node dissection is considered to be the treatment of choice for patients with muscle invasive bladder cancer (MIBC). Despite this aggressive treatment the outcome is poor and ultimately, 30% of the patients with ≥pT3 tumors develop a pelvic recurrence. We hypothesize that postoperative adjuvant external beam radiotherapy (EBRT) might prevent local and lymph node recurrence and improve disease free- and overall survival as loco-regional recurrence is linked to the development of distant metastasis.

Methods

We plan to perform a multicentric prospective phase two study including 76 patients. Eligible patients are patients with MIBC, treated with radical cystectomy and presenting with ≥1 of the following characteristics:

Pathological (p)T3 stage + presence of lymphovascular invasion on pathological examination

pT4 stage

<10 lymph nodes removed

positive lymph nodes

positive surgical margins

Patients will have a ¹⁸F–FDG PET-CT to rule out the presence of distant metastasis prior to EBRT. A median dose of 50 Gy in 25 fractions is prescribed to the pelvic lymph node regions with inclusion of the operative bladder bed in case of a positive surgical margin. Patients with suspected lymph nodes on PET- CT can still be included in the trial, but a simultaneous integrated boost to 74Gy to the positive lymph nodes will be delivered. Blood and urine samples will be collected on day-1 and last day of EBRT for evaluation of biomarkers. The primary endpoint is evaluation of acute ≥Grade 3 intestinal or grade 4 urinary toxicity, in case of a neo-bladder reconstruction, within 12 weeks after EBRT. Secondary endpoints are: assessment of QOL, late RTOG toxicity, local control, disease free survival and overall survival. Biomarkers in urine and blood will be correlated with secondary survival endpoints.

Discussion

This is a prospective phase 2 trial re-assessing the feasibility of adjuvant radiotherapy in high-risk MIBC.

Trial registration

The Ethics committee of the Ghent University Hospital (EC2014/0630) approved this study on 31/07/2014.

Trial registration on Clinicaltrials.gov (NCT02397434) on November 19, 2014.

http://ift.tt/2qCFkzN

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways

Abstract

Background

Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma.

Methods

We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53.

Results

Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells.

Conclusion

These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

http://ift.tt/2qCFiYH

Surfactant protein-D predicts prognosis of interstitial lung disease induced by anticancer agents in advanced lung cancer: a case control study

Abstract

Background

Interstitial lung diseases induced by anticancer agents (ILD-AA) are rare adverse effects of anticancer therapy. However, prognostic biomarkers for ILD-AA have not been identified in patients with advanced lung cancer. Our aim was to analyze the association between serum biomarkers sialylated carbohydrate antigen Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), and clinical characteristics in patients diagnosed with ILD-AA.

Methods

Between April 2011 and March 2016, 1224 advanced lung cancer patients received cytotoxic agents and epidermal growth factor receptor tyrosine kinase inhibitors at Juntendo University Hospital and Juntendo University Urayasu Hospital. Of these patients, those diagnosed with ILD-AA were enrolled in this case control study. ΔKL-6 and ΔSP-D were defined as the difference between the levels at the onset of ILD-AA and their respective levels prior to development of ILD-AA. We evaluated KL-6 and SP-D at the onset of ILD-AA, ΔKL-6 and ΔSP-D, the risk factors for death related to ILD-AA, the chest high resolution computed tomography (HRCT) findings, and survival time in patients diagnosed with ILD-AA.

Results

Thirty-six patients diagnosed with ILD-AA were enrolled in this study. Among them, 14 patients died of ILD-AA. ΔSP-D in the patients who died was significantly higher than that in the patients who survived. However, ΔKL-6 did not differ significantly between the two groups. Moreover, ΔSP-D in patients who exhibited diffuse alveolar damage was significantly higher than that in the other patterns on HRCT. Receiver operating characteristic curve analysis was used to set the optimal cut off value for ΔSP-D at 398 ng/mL. Survival time for patients with high ΔSP-D (≥ 398 ng/mL) was significantly shorter than that for patients with low ΔSP-D. Multivariate analysis revealed that ΔSP-D was a significant prognostic factor of ILD-AA.

Conclusions

This is the first research to evaluate high ΔSP-D (≥ 398 ng/mL) in patients with ILD-AA and to determine the risk factors for ILD-AA in advanced lung cancer patients. ΔSP-D might be a serum prognostic biomarker of ILD-AA. Clinicians should evaluate serum SP-D during chemotherapy and should carefully monitor the clinical course in patients with high ΔSP-D.

http://ift.tt/2pWAO2R

MicroRNA-30a regulates cell proliferation and tumor growth of colorectal cancer by targeting CD73

Abstract

Background

MicroRNAs are non-coding RNAs which regulate a variety of cellular functions in the development of tumors. Among the numerous microRNAs, microRNA-30a (miR-30a) is thought to play an important role in the processes of various human tumors. In this study, we aimed to explore the role of miR-30a in the process of colorectal cancer (CRC).

Methods

The quantitative real-time PCR and western blot analysis were used to detect the expressions of miR-30a and CD73 in CRC cell lines and clinical tissues. The luciferase reporter assay was conducted to validate the association between miR-30a and CD73. The CCK-8, terminal deoxynucleotidyl transferase dUTP -biotin nick end labeling (TUNEL) assays and cell cycle flow cytometry were carried out to verify the biological functions of miR-30a in vitro. The nude mouse tumorigenicity experiment was used to clarify the biological role of miR-30a in vivo.

Results

The expression of miR-30a was significantly reduced in tumor cells and tissues of CRC. The proliferation ability of CRC cells was suppressed and the apoptosis of cells was promoted when miR-30a is over-regulated, however, the biological effects would be inverse since the miR-30a is down-regulated. CD73 is thought to be a target binding gene of miR-30a because miR-30a can bind directly to the 3′-UTR of CD73 mRNA, subsequently reducing its expression. The proliferation suppression of the CRC cells mediated by miR-30a could be rescued after up-regulating the expression of CD73.

Conclusions

MiR-30a plays an important role on regulating the cell proliferation and apoptosis, thus affecting the growth of the tumor in CRC. And it may participate in the disease process of CRC by regulating the expression of CD73.

http://ift.tt/2qCJ3gU

The repressive effect of miR-148a on Wnt/β-catenin signaling involved in Glabridin-induced anti-angiogenesis in human breast cancer cells

Abstract

Background

Glabridin (GLA), a major component extracted from licorice root, has anti-inflammatory and antioxidant activities, but few studies report its mechanism of inhibition of angiogenesis. This study was an extension of our previous work, which demonstrated that GLA suppressed angiogenesis in human breast cancer (MDA-MB-231 and Hs-578T) cells. Breast cancer is one of the most common malignant diseases in females worldwide, and the major cause of mortality is metastasis that is primarily attributed to angiogenesis. Thus, anti-angiogenesis has become a strategy for the treatment of breast cancer.

Methods

Cell viability of different concentration treatment groups were detected by Cell Counting Kit-8 assay. The expression of several related genes in the Wnt1 signaling pathway in MDA-MB-231 and Hs-578T cells treated with GLA were measured at both the transcription and translation levels using quantitative real-time PCR analyses and western blotting. Immunofluorescence assay analyzed the nuclear translocation of β-catenin. The microRNA-inhibitor was used to knockdown microRNA-148a (miR-148a) expression. Angiogenic potentials of breast cancer cells were analyzed by enzyme-linked immunosorbent assay (ELISA) and tube formation in vitro.

Results

GLA attenuated angiogenesis by the suppression of miR-148a-mediated Wnt/β-catenin signaling pathway in two human breast cancer cell lines (MDA-MB-231 and Hs-578T). GLA also upregulated the expression of miR-148a in a dose-dependent manner, miR-148a, which could directly target Wnt-3′-untranslated regions (UTRs), and decreased the expression of Wnt1, leading to β-catenin accumulation in the membranes from the cytoplasm and nucleus. Downregulation of miR-148a contributed to the reduction of GLA-induced suppression of the Wnt/β-catenin signaling pathway, the angiogenesis and vascular endothelial grow factor (VEGF) secretion.

Conclusions

Our study identified a molecular mechanism of the GLA inhibition of angiogenesis through the Wnt/β-catenin signaling pathway via miR-148a, suggesting that GLA could serve as an adjuvant chemotherapeutic agent for breast cancer.

http://ift.tt/2pWJF4D

The E2F4 prognostic signature predicts pathological response to neoadjuvant chemotherapy in breast cancer patients

Abstract

Background

Neoadjuvant chemotherapy is a key component of breast cancer treatment regimens and pathologic complete response to this therapy varies among patients. This is presumably due to differences in the molecular mechanisms that underlie each tumor's disease pathology. Developing genomic clinical assays that accurately categorize responders from non-responders can provide patients with the most effective therapy for their individual disease.

Methods

We applied our previously developed E2F4 genomic signature to predict neoadjuvant chemotherapy response in breast cancer. E2F4 individual regulatory activity scores were calculated for 1129 patient samples across 5 independent breast cancer neoadjuvant chemotherapy datasets. Accuracy of the E2F4 signature in predicting neoadjuvant chemotherapy response was compared to that of the Oncotype DX and MammaPrint predictive signatures.

Results

In all datasets, E2F4 activity level was an accurate predictor of neoadjuvant chemotherapy response, with high E2F4 scores predictive of achieving pathologic complete response and low scores predictive of residual disease. These results remained significant even after stratifying patients by estrogen receptor (ER) status, tumor stage, and breast cancer molecular subtypes. Compared to the Oncotype DX and MammaPrint signatures, our E2F4 signature achieved similar performance in predicting neoadjuvant chemotherapy response, though all signatures performed better in ER+ tumors compared to ER- ones. The accuracy of our signature was reproducible across datasets and was maintained when refined from a 199-gene signature down to a clinic-friendly 33-gene panel.

Conclusion

Overall, we show that our E2F4 signature is accurate in predicting patient response to neoadjuvant chemotherapy. As this signature is more refined and comparable in performance to other clinically available gene expression assays in the prediction of neoadjuvant chemotherapy response, it should be considered when evaluating potential treatment options.

http://ift.tt/2qCUOnA

Immunohistochemical evaluation of epithelial ovarian carcinomas identifies three different expression patterns of the MX35 antigen, NaPi2b

Abstract

Background

To characterize the expression of the membrane transporter NaPi2b and antigen targeted by the MX35 antibody in ovarian tumor samples. The current interest to develop monoclonal antibody based therapy of ovarian cancer by targeting NaPi2b emphasizes the need for detailed knowledge and characterization of the expression pattern of this protein. For the majority of patients with ovarian carcinoma the risk of being diagnosed in late stages with extensive loco-regional spread disease is substantial, which stresses the need to develop improved therapeutic agents.

Methods

The gene and protein expression of SLC34A2/NaPi2b were analyzed in ovarian carcinoma tissues by QPCR (n = 73) and immunohistochemistry (n = 136). The expression levels and antigen localization were established and compared to the tumor characteristics and clinical data.

Results

Positive staining for the target protein, NaPi2b was detected for 93% of the malignant samples, and we identified three separate distribution patterns of the antigen within the tumors, based on the localization of NaPi2b. There were differences in the staining intensity as well as the distribution pattern when comparing the tumor grade and histology, the mucinous tumors presented a significantly lower expression of both the targeted protein and its related gene.

Conclusion

Our study identified differences regarding the level of the antigen expression between tumor grade and histology. We have identified differences in the antigen localization between borderline tumors, type 1 and type 2 tumors, and suggest that a pathological evaluation of NaPi2b in the tumors would be helpful in order to know which patients that would benefit from this targeted therapy.

http://ift.tt/2pWVJmv

Hepatitis B virus infection and active replication promote the formation of vascular invasion in hepatocellular carcinoma

Abstract

Background

Vascular invasion, including microvascular invasion (MVI) and portal vein tumor thrombus (PVTT), is associated with the postoperative recurrence of hepatocellular carcinoma (HCC). We aimed to investigate the potential impact of hepatitis B virus (HBV) activity on the development of vascular invasion.

Methods

Patients with HBV and tumor-related factors of HCC who had undergone hepatectomy were retrospectively enrolled and analyzed to identify the risk factors for developing vascular invasion.

Results

A total of 486 patients were included in this study. The overall proportion of patients with vascular invasion, including MVI and PVTT, was 60.3% (293/486). The incidence of MVI was 58.2% (283/486) whereas PVTT was 22.2% (108/486). Univariate analysis revealed that positive Hepatitis B virus surface Antigen (HBsAg) was significantly associated with the presence of vascular invasion. In a multivariate regression analysis carried out in patients with HBV-related HCC, positive Hepatitis B virus e Antigen (HBeAg)(OR = 1.83, P = 0.019) and a detectable seral HBV DNA load (OR = 1.68, P = 0.027) were independent risk factors of vascular invasion. The patients in the severe MVI group had a significantly higher rate of positive seral HBsAg (P = 0.005), positive seral HBeAg (P = 0.016), a detectable seral HBV DNA load (> 50 IU/ml) (P < 0.001) and a lower rate of anti-viral treatment (P = 0.002) compared with those in the mild MVI group and MVI-negative group. Whereas, HCC with PVTT invading the main trunk showed a significantly higher rate of positive HBsAg (P = 0.007), positive HBeAg (P = 0.04), cirrhosis (P = 0.005) and a lower rate of receiving antiviral treatment (P = 0.009) compared with patients with no PVTT or PVTT invading the ipsilateral portal vein. Patients with vascular invasion also had a significantly higher level of seral HBV DNA load than patients without vascular invasion (P = 0.008).

Conclusions

In HCC patients, HBV infection and active HBV replication were associated with the development of vascular invasion.

http://ift.tt/2qCYghV

Hepatitis B virus infection and active replication promote the formation of vascular invasion in hepatocellular carcinoma

Abstract

Background

Vascular invasion, including microvascular invasion (MVI) and portal vein tumor thrombus (PVTT), is associated with the postoperative recurrence of hepatocellular carcinoma (HCC). We aimed to investigate the potential impact of hepatitis B virus (HBV) activity on the development of vascular invasion.

Methods

Patients with HBV and tumor-related factors of HCC who had undergone hepatectomy were retrospectively enrolled and analyzed to identify the risk factors for developing vascular invasion.

Results

A total of 486 patients were included in this study. The overall proportion of patients with vascular invasion, including MVI and PVTT, was 60.3% (293/486). The incidence of MVI was 58.2% (283/486) whereas PVTT was 22.2% (108/486). Univariate analysis revealed that positive Hepatitis B virus surface Antigen (HBsAg) was significantly associated with the presence of vascular invasion. In a multivariate regression analysis carried out in patients with HBV-related HCC, positive Hepatitis B virus e Antigen (HBeAg)(OR = 1.83, P = 0.019) and a detectable seral HBV DNA load (OR = 1.68, P = 0.027) were independent risk factors of vascular invasion. The patients in the severe MVI group had a significantly higher rate of positive seral HBsAg (P = 0.005), positive seral HBeAg (P = 0.016), a detectable seral HBV DNA load (> 50 IU/ml) (P < 0.001) and a lower rate of anti-viral treatment (P = 0.002) compared with those in the mild MVI group and MVI-negative group. Whereas, HCC with PVTT invading the main trunk showed a significantly higher rate of positive HBsAg (P = 0.007), positive HBeAg (P = 0.04), cirrhosis (P = 0.005) and a lower rate of receiving antiviral treatment (P = 0.009) compared with patients with no PVTT or PVTT invading the ipsilateral portal vein. Patients with vascular invasion also had a significantly higher level of seral HBV DNA load than patients without vascular invasion (P = 0.008).

Conclusions

In HCC patients, HBV infection and active HBV replication were associated with the development of vascular invasion.

from Cancer via ola Kala on Inoreader http://ift.tt/2qCYghV
via IFTTT

Adjuvant radiotherapy after radical cystectomy for patients with muscle invasive bladder cancer: a phase II trial

Abstract

Background

Neo-adjuvant chemotherapy followed by radical cystectomy with extended pelvic lymph node dissection is considered to be the treatment of choice for patients with muscle invasive bladder cancer (MIBC). Despite this aggressive treatment the outcome is poor and ultimately, 30% of the patients with ≥pT3 tumors develop a pelvic recurrence. We hypothesize that postoperative adjuvant external beam radiotherapy (EBRT) might prevent local and lymph node recurrence and improve disease free- and overall survival as loco-regional recurrence is linked to the development of distant metastasis.

Methods

We plan to perform a multicentric prospective phase two study including 76 patients. Eligible patients are patients with MIBC, treated with radical cystectomy and presenting with ≥1 of the following characteristics:

Pathological (p)T3 stage + presence of lymphovascular invasion on pathological examination

pT4 stage

<10 lymph nodes removed

positive lymph nodes

positive surgical margins

Patients will have a ¹⁸F–FDG PET-CT to rule out the presence of distant metastasis prior to EBRT. A median dose of 50 Gy in 25 fractions is prescribed to the pelvic lymph node regions with inclusion of the operative bladder bed in case of a positive surgical margin. Patients with suspected lymph nodes on PET- CT can still be included in the trial, but a simultaneous integrated boost to 74Gy to the positive lymph nodes will be delivered. Blood and urine samples will be collected on day-1 and last day of EBRT for evaluation of biomarkers. The primary endpoint is evaluation of acute ≥Grade 3 intestinal or grade 4 urinary toxicity, in case of a neo-bladder reconstruction, within 12 weeks after EBRT. Secondary endpoints are: assessment of QOL, late RTOG toxicity, local control, disease free survival and overall survival. Biomarkers in urine and blood will be correlated with secondary survival endpoints.

Discussion

This is a prospective phase 2 trial re-assessing the feasibility of adjuvant radiotherapy in high-risk MIBC.

Trial registration

The Ethics committee of the Ghent University Hospital (EC2014/0630) approved this study on 31/07/2014.

Trial registration on Clinicaltrials.gov (NCT02397434) on November 19, 2014.

from Cancer via ola Kala on Inoreader http://ift.tt/2qCFkzN
via IFTTT

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways

Abstract

Background

Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma.

Methods

We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53.

Results

Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells.

Conclusion

These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

from Cancer via ola Kala on Inoreader http://ift.tt/2qCFiYH
via IFTTT

Surfactant protein-D predicts prognosis of interstitial lung disease induced by anticancer agents in advanced lung cancer: a case control study

Abstract

Background

Interstitial lung diseases induced by anticancer agents (ILD-AA) are rare adverse effects of anticancer therapy. However, prognostic biomarkers for ILD-AA have not been identified in patients with advanced lung cancer. Our aim was to analyze the association between serum biomarkers sialylated carbohydrate antigen Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D), and clinical characteristics in patients diagnosed with ILD-AA.

Methods

Between April 2011 and March 2016, 1224 advanced lung cancer patients received cytotoxic agents and epidermal growth factor receptor tyrosine kinase inhibitors at Juntendo University Hospital and Juntendo University Urayasu Hospital. Of these patients, those diagnosed with ILD-AA were enrolled in this case control study. ΔKL-6 and ΔSP-D were defined as the difference between the levels at the onset of ILD-AA and their respective levels prior to development of ILD-AA. We evaluated KL-6 and SP-D at the onset of ILD-AA, ΔKL-6 and ΔSP-D, the risk factors for death related to ILD-AA, the chest high resolution computed tomography (HRCT) findings, and survival time in patients diagnosed with ILD-AA.

Results

Thirty-six patients diagnosed with ILD-AA were enrolled in this study. Among them, 14 patients died of ILD-AA. ΔSP-D in the patients who died was significantly higher than that in the patients who survived. However, ΔKL-6 did not differ significantly between the two groups. Moreover, ΔSP-D in patients who exhibited diffuse alveolar damage was significantly higher than that in the other patterns on HRCT. Receiver operating characteristic curve analysis was used to set the optimal cut off value for ΔSP-D at 398 ng/mL. Survival time for patients with high ΔSP-D (≥ 398 ng/mL) was significantly shorter than that for patients with low ΔSP-D. Multivariate analysis revealed that ΔSP-D was a significant prognostic factor of ILD-AA.

Conclusions

This is the first research to evaluate high ΔSP-D (≥ 398 ng/mL) in patients with ILD-AA and to determine the risk factors for ILD-AA in advanced lung cancer patients. ΔSP-D might be a serum prognostic biomarker of ILD-AA. Clinicians should evaluate serum SP-D during chemotherapy and should carefully monitor the clinical course in patients with high ΔSP-D.

from Cancer via ola Kala on Inoreader http://ift.tt/2pWAO2R
via IFTTT

MicroRNA-30a regulates cell proliferation and tumor growth of colorectal cancer by targeting CD73

Abstract

Background

MicroRNAs are non-coding RNAs which regulate a variety of cellular functions in the development of tumors. Among the numerous microRNAs, microRNA-30a (miR-30a) is thought to play an important role in the processes of various human tumors. In this study, we aimed to explore the role of miR-30a in the process of colorectal cancer (CRC).

Methods

The quantitative real-time PCR and western blot analysis were used to detect the expressions of miR-30a and CD73 in CRC cell lines and clinical tissues. The luciferase reporter assay was conducted to validate the association between miR-30a and CD73. The CCK-8, terminal deoxynucleotidyl transferase dUTP -biotin nick end labeling (TUNEL) assays and cell cycle flow cytometry were carried out to verify the biological functions of miR-30a in vitro. The nude mouse tumorigenicity experiment was used to clarify the biological role of miR-30a in vivo.

Results

The expression of miR-30a was significantly reduced in tumor cells and tissues of CRC. The proliferation ability of CRC cells was suppressed and the apoptosis of cells was promoted when miR-30a is over-regulated, however, the biological effects would be inverse since the miR-30a is down-regulated. CD73 is thought to be a target binding gene of miR-30a because miR-30a can bind directly to the 3′-UTR of CD73 mRNA, subsequently reducing its expression. The proliferation suppression of the CRC cells mediated by miR-30a could be rescued after up-regulating the expression of CD73.

Conclusions

MiR-30a plays an important role on regulating the cell proliferation and apoptosis, thus affecting the growth of the tumor in CRC. And it may participate in the disease process of CRC by regulating the expression of CD73.

from Cancer via ola Kala on Inoreader http://ift.tt/2qCJ3gU
via IFTTT

The repressive effect of miR-148a on Wnt/β-catenin signaling involved in Glabridin-induced anti-angiogenesis in human breast cancer cells

Abstract

Background

Glabridin (GLA), a major component extracted from licorice root, has anti-inflammatory and antioxidant activities, but few studies report its mechanism of inhibition of angiogenesis. This study was an extension of our previous work, which demonstrated that GLA suppressed angiogenesis in human breast cancer (MDA-MB-231 and Hs-578T) cells. Breast cancer is one of the most common malignant diseases in females worldwide, and the major cause of mortality is metastasis that is primarily attributed to angiogenesis. Thus, anti-angiogenesis has become a strategy for the treatment of breast cancer.

Methods

Cell viability of different concentration treatment groups were detected by Cell Counting Kit-8 assay. The expression of several related genes in the Wnt1 signaling pathway in MDA-MB-231 and Hs-578T cells treated with GLA were measured at both the transcription and translation levels using quantitative real-time PCR analyses and western blotting. Immunofluorescence assay analyzed the nuclear translocation of β-catenin. The microRNA-inhibitor was used to knockdown microRNA-148a (miR-148a) expression. Angiogenic potentials of breast cancer cells were analyzed by enzyme-linked immunosorbent assay (ELISA) and tube formation in vitro.

Results

GLA attenuated angiogenesis by the suppression of miR-148a-mediated Wnt/β-catenin signaling pathway in two human breast cancer cell lines (MDA-MB-231 and Hs-578T). GLA also upregulated the expression of miR-148a in a dose-dependent manner, miR-148a, which could directly target Wnt-3′-untranslated regions (UTRs), and decreased the expression of Wnt1, leading to β-catenin accumulation in the membranes from the cytoplasm and nucleus. Downregulation of miR-148a contributed to the reduction of GLA-induced suppression of the Wnt/β-catenin signaling pathway, the angiogenesis and vascular endothelial grow factor (VEGF) secretion.

Conclusions

Our study identified a molecular mechanism of the GLA inhibition of angiogenesis through the Wnt/β-catenin signaling pathway via miR-148a, suggesting that GLA could serve as an adjuvant chemotherapeutic agent for breast cancer.

from Cancer via ola Kala on Inoreader http://ift.tt/2pWJF4D
via IFTTT

The E2F4 prognostic signature predicts pathological response to neoadjuvant chemotherapy in breast cancer patients

Abstract

Background

Neoadjuvant chemotherapy is a key component of breast cancer treatment regimens and pathologic complete response to this therapy varies among patients. This is presumably due to differences in the molecular mechanisms that underlie each tumor's disease pathology. Developing genomic clinical assays that accurately categorize responders from non-responders can provide patients with the most effective therapy for their individual disease.

Methods

We applied our previously developed E2F4 genomic signature to predict neoadjuvant chemotherapy response in breast cancer. E2F4 individual regulatory activity scores were calculated for 1129 patient samples across 5 independent breast cancer neoadjuvant chemotherapy datasets. Accuracy of the E2F4 signature in predicting neoadjuvant chemotherapy response was compared to that of the Oncotype DX and MammaPrint predictive signatures.

Results

In all datasets, E2F4 activity level was an accurate predictor of neoadjuvant chemotherapy response, with high E2F4 scores predictive of achieving pathologic complete response and low scores predictive of residual disease. These results remained significant even after stratifying patients by estrogen receptor (ER) status, tumor stage, and breast cancer molecular subtypes. Compared to the Oncotype DX and MammaPrint signatures, our E2F4 signature achieved similar performance in predicting neoadjuvant chemotherapy response, though all signatures performed better in ER+ tumors compared to ER- ones. The accuracy of our signature was reproducible across datasets and was maintained when refined from a 199-gene signature down to a clinic-friendly 33-gene panel.

Conclusion

Overall, we show that our E2F4 signature is accurate in predicting patient response to neoadjuvant chemotherapy. As this signature is more refined and comparable in performance to other clinically available gene expression assays in the prediction of neoadjuvant chemotherapy response, it should be considered when evaluating potential treatment options.

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Immunohistochemical evaluation of epithelial ovarian carcinomas identifies three different expression patterns of the MX35 antigen, NaPi2b

Abstract

Background

To characterize the expression of the membrane transporter NaPi2b and antigen targeted by the MX35 antibody in ovarian tumor samples. The current interest to develop monoclonal antibody based therapy of ovarian cancer by targeting NaPi2b emphasizes the need for detailed knowledge and characterization of the expression pattern of this protein. For the majority of patients with ovarian carcinoma the risk of being diagnosed in late stages with extensive loco-regional spread disease is substantial, which stresses the need to develop improved therapeutic agents.

Methods

The gene and protein expression of SLC34A2/NaPi2b were analyzed in ovarian carcinoma tissues by QPCR (n = 73) and immunohistochemistry (n = 136). The expression levels and antigen localization were established and compared to the tumor characteristics and clinical data.

Results

Positive staining for the target protein, NaPi2b was detected for 93% of the malignant samples, and we identified three separate distribution patterns of the antigen within the tumors, based on the localization of NaPi2b. There were differences in the staining intensity as well as the distribution pattern when comparing the tumor grade and histology, the mucinous tumors presented a significantly lower expression of both the targeted protein and its related gene.

Conclusion

Our study identified differences regarding the level of the antigen expression between tumor grade and histology. We have identified differences in the antigen localization between borderline tumors, type 1 and type 2 tumors, and suggest that a pathological evaluation of NaPi2b in the tumors would be helpful in order to know which patients that would benefit from this targeted therapy.

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Trunk rotation,Cervical induced horizontal nystagmus,“neck torsion test” (NTT),There is a “cervicotonic provocation nystagmus” in response to objective reaction to cervical straining with a static trunk excursion.

http://otorhinolaryngology-crete.blogspot.gr/2017/05/cervico-vestibular-muscle-afferents.html

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com

Prolonged endotracheal intubation is the main indication of tracheostomy, performed after two weeks of intubation. Although there were no major early complications, laryngotracheal stenosis is still a challenging sequel for tracheostomy that needs to be investigated to be prevented.

http://otorhinolaryngology-crete.blogspot.com/2017/05/prolonged-endotracheal-intubation-is.html

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com

Characterization of the Anti-PD-1 Antibody REGN2810 and Its Antitumor Activity in Human PD-1 Knock-In Mice

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti–PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1–dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861–70. ©2017 AACR.

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Acquired Resistance to the Hsp90 Inhibitor, Ganetespib, in KRAS-Mutant NSCLC Is Mediated via Reactivation of the ERK-p90RSK-mTOR Signaling Network

Approximately 25% of non–small cell lung cancer (NSCLC) patients have KRAS mutations, and no effective therapeutic strategy exists for these patients. The use of Hsp90 inhibitors in KRAS-mutant NSCLC appeared to be a promising approach, as these inhibitors target many KRAS downstream effectors; however, limited clinical efficacy has been observed due to resistance. Here, we examined the mechanism(s) of acquired resistance to the Hsp90 inhibitor, ganetespib, and identified novel and rationally devised Hsp90 inhibitor combinations, which may prevent and overcome resistance to Hsp90 inhibitors. We derived KRAS-mutant NSCLC ganetespib-resistant cell lines to identify the resistance mechanism(s) and identified hyperactivation of RAF/MEK/ERK/RSK and PI3K/AKT/mTOR pathways as key resistance mechanisms. Furthermore, we found that ganetespib-resistant cells are "addicted" to these pathways, as ganetespib resistance leads to synthetic lethality to a dual PI3K/mTOR, a PI3K, or an ERK inhibitor. Interestingly, the levels and activity of a key activator of the mTOR pathway and an ERK downstream target, p90 ribosomal S6 kinase (RSK), were also increased in the ganetespib-resistant cells. Genetic or pharmacologic inhibition of p90RSK in ganetespib-resistant cells restored sensitivity to ganetespib, whereas p90RSK overexpression induced ganetespib resistance in naïve cells, validating p90RSK as a mediator of resistance and a novel therapeutic target. Our studies offer a way forward for Hsp90 inhibitors through the rational design of Hsp90 inhibitor combinations that may prevent and/or overcome resistance to Hsp90 inhibitors, providing an effective therapeutic strategy for KRAS-mutant NSCLC. Mol Cancer Ther; 16(5); 793–804. ©2017 AACR.

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Synthesis and Evaluation of the Novel Prostamide, 15-Deoxy, {Delta}12,14-Prostamide J2, as a Selective Antitumor Therapeutic

15-deoxy, ^12,14-prostaglandin J₂-ethanolamide, also known as 15-deoxy, ^12,14-prostamide J₂ (15d-PMJ₂) is a novel product of the metabolism of arachidonoyl ethanolamide (AEA) by COX-2. 15d-PMJ₂ preferentially induced cell death and apoptosis in tumorigenic A431 keratinocytes and B16F10 melanoma cells compared with nontumorigenic HaCaT keratinocytes and Melan-A melanocytes. Activation of the ER stress execution proteins, PERK and CHOP10, was evaluated to determine whether this process was involved in 15d-PMJ₂ cell death. 15d-PMJ₂ increased the phosphorylation of PERK and expression of CHOP10 in tumorigenic but not nontumorigenic cells. The known ER stress inhibitors, salubrinal and 4-phenylbutaric acid, significantly inhibited 15d-PMJ₂–mediated apoptosis, suggesting ER stress as a primary apoptotic mediator. Furthermore, the reactive double bond present within the cyclopentenone structure of 15d-PMJ₂ was identified as a required moiety for the induction of ER stress apoptosis. The effect of 15d-PMJ₂ on B16F10 melanoma growth was also evaluated by dosing C57BL/6 mice with 0.5 mg/kg 15d-PMJ₂. Tumors of animals treated with 15d-PMJ₂ exhibited significantly reduced growth and mean weights compared with vehicle and untreated animals. TUNEL and IHC analysis of tumor tissues showed significant cell death and ER stress in tumors of 15d-PMJ₂–treated compared with control group animals. Taken together, these findings suggest that the novel prostamide, 15d-PMJ₂, possesses potent antitumor activity in vitro and in vivo. Mol Cancer Ther; 16(5); 838–49. ©2017 AACR.

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Hey Factors at the Crossroad of Tumorigenesis and Clinical Therapeutic Modulation of Hey for Anticancer Treatment

Hairy and Enhancer-of-split related with YRPW motif (Hey) transcription factors are important regulators of stem cell embryogenesis. Clinical relevance shows that they are also highly expressed in malignant carcinoma. Recent studies have highlighted functions for the Hey factors in tumor metastasis, the maintenance of cancer cell self-renewal, as well as proliferation and the promotion of tumor angiogenesis. Pathways that regulate Hey gene expression, such as Notch and TGFβ signaling, are frequently aberrant in numerous cancers. In addition, Hey factors control downstream targets via recruitment of histone deacetylases (HDAC). Targeting these signaling pathways or HDACs may reverse tumor progression and provide clinical benefit for cancer patients. Thus, some small molecular inhibitors or monoclonal antibodies of each of these signaling pathways have been studied in clinical trials. This review focuses on the involvement of Hey proteins in malignant carcinoma progression and provides valuable therapeutic information for anticancer treatment. Mol Cancer Ther; 16(5); 775–86. ©2017 AACR.

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Dual Targeting of Epithelial Ovarian Cancer Via Folate Receptor {alpha} and the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates

Folate uptake in epithelial ovarian cancer (EOC) involves the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT), both facilitative transporters and folate receptor (FR) α. Although in primary EOC specimens, FRα is widely expressed and increases with tumor stage, PCFT was expressed independent of tumor stage (by real-time RT-PCR and IHC). EOC cell line models, including cisplatin sensitive (IGROV1 and A2780) and resistant (SKOV3 and TOV112D) cells, expressed a 17-fold range of FRα and similar amounts (within ~2-fold) of PCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates AGF94 and AGF154 exhibited potent antiproliferative activities toward all of the EOC cell lines, reflecting selective cellular uptake by FRα and/or PCFT over RFC. When IGROV1 cells were pretreated with AGF94 at pH 6.8, clonogenicity was potently inhibited, confirming cell killing. FRα was knocked down in IGROV1 cells with lentiviral shRNAs. Two FRα knockdown clones (KD-4 and KD-10) showed markedly reduced binding and uptake of [³H]folic acid and [³H]AGF154 by FRα, but maintained high levels of [³H]AGF154 uptake by PCFT compared to nontargeted control cells. In proliferation assays, KD-4 and KD-10 cells preserved in vitro inhibition by AGF94 and AGF154, compared to a nontargeted control, attributable to residual FRα- and substantial PCFT-mediated uptake. KD-10 tumor xenografts in severe-compromised immune-deficient mice were likewise sensitive to AGF94. Collectively, our results demonstrate the substantial therapeutic potential of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with dual targeting of PCFT and FRα toward EOCs that express a range of FRα, along with PCFT, as well as cisplatin resistance. Mol Cancer Ther; 16(5); 819–30. ©2017 AACR.

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Nodal Signaling as a Developmental Therapeutics Target in Oncology

The tumor microenvironment is a vital feature of oncogenesis and tumor progression. There are several parallels between cancer cells and early developmental stem cells, including their plasticity and signaling mechanisms. In early fetal development, Nodal is expressed for endodermal and mesodermal differentiation. This expression has been shown reemerge in the setting of epithelial cancers, such as breast and melanoma. High Nodal expression correlates to an aggressive tumor grade in these malignancies. Nodal signal begins with its interaction with its coreceptor, Cripto-1, leading to activation of Smad2/Smad3 and ultimately downstream transcription and translation. Lefty is the natural inhibitor of Nodal and controls Nodal signaling during fetal development. However, cancer cells lack the presence of Lefty, thus leading to uncontrolled tumor growth. Given this understanding, inhibition of the Nodal pathway offers a new novel therapeutic target in oncology. Mol Cancer Ther; 16(5); 787–92. ©2017 AACR.

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Fluorinated N,N'-Diarylureas As Novel Therapeutic Agents Against Cancer Stem Cells

Colorectal cancer is the second-leading cause of cancer-related mortality in the United States. More than 50% of patients with colorectal cancer will develop local recurrence or distant organ metastasis. Cancer stem cells play a major role in the survival and metastasis of cancer cells. In this study, we examined the effects of novel AMP-activated protein kinase (AMPK) activating compounds on colorectal cancer metastatic and stem cell lines as potential candidates for chemotherapy. We found that activation of AMPK by all fluorinated N,N-diarylureas (FND) compounds at micromolar levels significantly inhibited the cell-cycle progression and subsequent cellular proliferation. In addition, we demonstrated that select FNDs significantly increased apoptosis in colorectal cancer metastatic and cancer stem cells. Therefore, FNDs hold considerable promise in the treatment of metastatic colorectal cancer, through elimination of both regular cancer cells and cancer stem cells. Mol Cancer Ther; 16(5); 831–7. ©2017 AACR.

http://ift.tt/2p4iUGp

Characterization of a Dual Rac/Cdc42 Inhibitor MBQ-167 in Metastatic Cancer

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC₅₀, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo. Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC₅₀ values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI₅₀ of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167–mediated G₂–M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10x more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805–18. ©2017 AACR.

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Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

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Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody–drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871–8. ©2017 AACR.

http://ift.tt/2pGBqbS

Preclinical Antitumor Efficacy of BAY 1129980--a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non–small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody–drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893–904. ©2017 AACR.

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Inhibition of Isoprenylcysteine Carboxylmethyltransferase Induces Cell-Cycle Arrest and Apoptosis through p21 and p21-Regulated BNIP3 Induction in Pancreatic Cancer

Pancreatic cancer remains one of the most difficult to treat human cancers despite recent advances in targeted therapy. Inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that posttranslationally modifies a group of proteins including several small GTPases, suppresses proliferation of some human cancer cells. However, the efficacy of ICMT inhibition on human pancreatic cancer has not been evaluated. In this study, we have evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition. In these cells, ICMT suppression inhibited proliferation and induced apoptosis. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both a small-molecule inhibitor and shRNA-targeting ICMT. Mechanistically, we found that, in sensitive pancreatic cancer cells, ICMT inhibition induced mitochondrial respiratory deficiency and cellular energy depletion, leading to significant upregulation of p21. Furthermore, we characterized the role of p21 as a regulator and coordinator of cell signaling that responds to cell energy depletion. Apoptosis, but not autophagy, that is induced via p21-activated BNIP3 expression accounts for the efficacy of ICMT inhibition in sensitive pancreatic cancer cells in both in vitro and in vivo models. In contrast, cells resistant to ICMT inhibition demonstrated no mitochondria dysfunction or p21 signaling changes under ICMT suppression. These findings not only identify pancreatic cancers as potential therapeutic targets for ICMT suppression but also provide an avenue for identifying those subtypes that would be most responsive to agents targeting this critical enzyme. Mol Cancer Ther; 16(5); 914–23. ©2017 AACR.

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Highly Potent, Anthracycline-based Antibody-Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

Antibody–drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required. Mol Cancer Ther; 16(5); 879–92. ©2017 AACR.

http://ift.tt/2p4ivnk

Lipid Nanoparticle-Mediated Delivery of Anti-miR-17 Family Oligonucleotide Suppresses Hepatocellular Carcinoma Growth

Hepatocellular carcinoma (HCC) is one of the most common human malignancies with poor prognosis and urgent unmet medical need. Aberrant expression of multiple members of the miR-17 family are frequently observed in HCC, and their overexpression promotes tumorigenic properties of HCC cells. However, whether pharmacologic inhibition of the miR-17 family inhibits HCC growth remains unknown. In this study, we validated that the miR-17 family was upregulated in a subset of HCC tumors and cell lines and its inhibition by a tough decoy inhibitor suppressed the growth of Hep3B and HepG2 cells, which overexpress the miR-17 family. Furthermore, inhibition of the miR-17 family led to a global derepression of direct targets of the family in all three HCC cell lines tested. Pathway analysis of the deregulated genes indicated that the genes associated with TGFβ signaling pathway were highly enriched in Hep3B and HepG2 cells. A miR-17 family target gene signature was established and used to identify RL01-17(5), a lipid nanoparticle encapsulating a potent anti-miR-17 family oligonucleotide. To address whether pharmacologic modulation of the miR-17 family can inhibit HCC growth, RL01-17(5) was systemically administrated to orthotopic Hep3B xenografts. Suppression of Hep3B tumor growth in vivo was observed and tumor growth inhibition correlated with induction of miR-17 family target genes. Together, this study provides proof-of-concept for targeting the miR-17 family in HCC therapy. Mol Cancer Ther; 16(5); 905–13. ©2017 AACR.

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Role of STAT3 and FOXO1 in the Divergent Therapeutic Responses of Non-metastatic and Metastatic Bladder Cancer Cells to miR-145

Although miR-145 is the most frequently downregulated miRNA in bladder cancer, its exact stage association and downstream effector have not been defined. Here, we found that miR-145 was upregulated in human patients with bladder cancer with lymph node metastasis and in metastatic T24T cell line. Forced expression of miR-145 promoted anchorage-independent growth of T24T cells accompanied by the downregulation of forkhead box class O1 (FOXO1). In contrast, in non-metastatic T24 cells, miR-145 overexpression inhibited cell growth with upregulation of FOXO1, and the knockdown of FOXO1 abolished the miR-145–mediated inhibition of cell growth. Mechanistic studies revealed that miR-145 directly bound to and attenuated 3'-untranslated region (UTR) activity of foxo1 mRNA in both T24 and T24T cells. Interestingly, miR-145 suppressed STAT3 phosphorylation at Tyr705 and increased foxo1 promoter transcriptional activity in T24 cells, but not in T24T cells, suggesting a role of STAT3 in the divergent responses to miR-145. Supporting this was our finding that STAT3 knockdown mimicked miR-145–mediated upregulation of FOXO1 in T24T cells and inhibition of anchorage-independent growth. Consistently, ectopic expression of miR-145 promoted tumor formation of xenograft T24T cells, whereas such promoting effect became inhibitory due to specific knockdown of STAT3. Together, our findings demonstrate the stage-specific association and function of miR-145 in bladder cancers and provide novel insights into the therapeutic targeting of miR-145. Mol Cancer Ther; 16(5); 924–35. ©2017 AACR.

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Calcium-Dependent Enhancement by Extracellular Acidity of the Cytotoxicity of Mitochondrial Inhibitors against Melanoma

Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Because acidity potentiates malignant phenotypes, therapeutic strategies that enhance the targeting of oncogenic mechanisms in an acidic microenvironment should be effective. We report here that drugs which abrogate mitochondrial respiration show enhanced cytotoxicity against melanoma cells in a normoxic but acidic extracellular pH, independent from P53 mutations, BRAF (V600E) mutations, and/or resistance against BRAF inhibitors. Conversely, the cytotoxicity against melanoma cells of mitochondrial inhibitors is impaired by a neutral or alkaline extracellular pH, and in vivo systemic alkalinization with NaHCO₃ enhanced subcutaneous tumor growth and lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin. Intracellular calcium (Ca²⁺) was significantly increased in melanoma cells treated with mitochondrial inhibitors at an acidic extracellular pH and an intracellular Ca²⁺ chelator, BAPTA/AM, inhibited cytoplasmic Ca²⁺ as well as melanoma cell death. Surprisingly, ROS scavengers synergized with increased apoptosis in cells treated with mitochondrial inhibitors, suggesting that ROS contributes to cell survival in this context. Notably, the cytotoxic enhancement of mitochondrial inhibitors by acidity was distinct from PGC1alpha-driven mitochondrial addiction, from therapy-induced senescence, and from slow, JARID1B-high–associated cell cycling, all of which have been shown to promote vulnerability to mitochondrial inhibition. These data indicate that extracellular pH profoundly modulates the cytotoxicity of mitochondrial inhibitors against cancer cells. Mol Cancer Ther; 16(5); 936–47. ©2017 AACR.

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Cell-Free DNA from Ascites and Pleural Effusions: Molecular Insights into Genomic Aberrations and Disease Biology

Collection of cell-free DNA (cfDNA) from the blood of individuals with cancer has permitted noninvasive tumor genome analysis. Detection and characterization of cfDNA in ascites and pleural effusions have not yet been reported. Herein, we analyzed cfDNA in the ascites and pleural effusions from six individuals with metastatic cancer. In all cases, cfDNA copy number variations (CNV) were discovered within the effusate. One individual had a relevant alteration with a high copy amplification in EGFR in a never smoker with lung cancer, who showed only MDM2 and CDK4 amplification in a prior tissue biopsy. Another subject with metastatic breast cancer had cytology-positive ascites and an activating PIK3CA mutation identified in the tissue, blood, and ascites collectively. This individual had tumor regression after the administration of the mTOR inhibitor everolimus and had evidence of chromotripsis from chromosomal rearrangements noted in the cell-free ascitic fluid. These results indicate that cfDNA from ascites and pleural effusions may provide additional information not detected with tumor and plasma cell-free DNA molecular characterization, and a context for important insights into tumor biology and clonal dynamic change within primary tumor and metastatic deposits. Mol Cancer Ther; 16(5); 948–55. ©2017 AACR.

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Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro. We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I–II study of 23 dogs with spontaneous, stage I–II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956–65. ©2017 AACR.

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HSP70 Inhibition Synergistically Enhances the Effects of Magnetic Fluid Hyperthermia in Ovarian Cancer

Hyperthermia has been investigated as a potential treatment for cancer. However, specificity in hyperthermia application remains a significant challenge. Magnetic fluid hyperthermia (MFH) may be an alternative to surpass such a challenge, but implications of MFH at the cellular level are not well understood. Therefore, the present work focused on the examination of gene expression after MFH treatment and using such information to identify target genes that when inhibited could produce an enhanced therapeutic outcome after MFH. Genomic analyzes were performed using ovarian cancer cells exposed to MFH for 30 minutes at 43°C, which revealed that heat shock protein (HSP) genes, including HSPA6, were upregulated. HSPA6 encodes the Hsp70, and its expression was confirmed by PCR in HeyA8 and A2780cp20 ovarian cancer cells. Two strategies were investigated to inhibit Hsp70-related genes, siRNA and Hsp70 protein function inhibition by 2-phenylethyenesulfonamide (PES). Both strategies resulted in decreased cell viability following exposure to MFH. Combination index was calculated for PES treatment reporting a synergistic effect. In vivo efficacy experiments with HSPA6 siRNA and MFH were performed using the A2780cp20 and HeyA8 ovarian cancer mouse models. A significantly reduction in tumor growth rate was observed with combination therapy. PES and MFH efficacy were also evaluated in the HeyA8 intraperitoneal tumor model, and resulted in robust antitumor effects. This work demonstrated that HSP70 inhibition combination with MFH generate a synergistic effect and could be a promising target to enhance MFH therapeutic outcomes in ovarian cancer. Mol Cancer Ther; 16(5); 966–76. ©2017 AACR.

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Retraction: Proteasome Inhibition Blocks NF-{kappa}B and ERK1/2 Pathways, Restores Antigen Expression, and Sensitizes Resistant Human Melanoma to TCR-Engineered CTLs

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Synthesis and Evaluation of the Novel Prostamide, 15-Deoxy, {Delta}12,14-Prostamide J2, as a Selective Antitumor Therapeutic

15-deoxy, ^12,14-prostaglandin J₂-ethanolamide, also known as 15-deoxy, ^12,14-prostamide J₂ (15d-PMJ₂) is a novel product of the metabolism of arachidonoyl ethanolamide (AEA) by COX-2. 15d-PMJ₂ preferentially induced cell death and apoptosis in tumorigenic A431 keratinocytes and B16F10 melanoma cells compared with nontumorigenic HaCaT keratinocytes and Melan-A melanocytes. Activation of the ER stress execution proteins, PERK and CHOP10, was evaluated to determine whether this process was involved in 15d-PMJ₂ cell death. 15d-PMJ₂ increased the phosphorylation of PERK and expression of CHOP10 in tumorigenic but not nontumorigenic cells. The known ER stress inhibitors, salubrinal and 4-phenylbutaric acid, significantly inhibited 15d-PMJ₂–mediated apoptosis, suggesting ER stress as a primary apoptotic mediator. Furthermore, the reactive double bond present within the cyclopentenone structure of 15d-PMJ₂ was identified as a required moiety for the induction of ER stress apoptosis. The effect of 15d-PMJ₂ on B16F10 melanoma growth was also evaluated by dosing C57BL/6 mice with 0.5 mg/kg 15d-PMJ₂. Tumors of animals treated with 15d-PMJ₂ exhibited significantly reduced growth and mean weights compared with vehicle and untreated animals. TUNEL and IHC analysis of tumor tissues showed significant cell death and ER stress in tumors of 15d-PMJ₂–treated compared with control group animals. Taken together, these findings suggest that the novel prostamide, 15d-PMJ₂, possesses potent antitumor activity in vitro and in vivo. Mol Cancer Ther; 16(5); 838–49. ©2017 AACR.

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Hey Factors at the Crossroad of Tumorigenesis and Clinical Therapeutic Modulation of Hey for Anticancer Treatment

Hairy and Enhancer-of-split related with YRPW motif (Hey) transcription factors are important regulators of stem cell embryogenesis. Clinical relevance shows that they are also highly expressed in malignant carcinoma. Recent studies have highlighted functions for the Hey factors in tumor metastasis, the maintenance of cancer cell self-renewal, as well as proliferation and the promotion of tumor angiogenesis. Pathways that regulate Hey gene expression, such as Notch and TGFβ signaling, are frequently aberrant in numerous cancers. In addition, Hey factors control downstream targets via recruitment of histone deacetylases (HDAC). Targeting these signaling pathways or HDACs may reverse tumor progression and provide clinical benefit for cancer patients. Thus, some small molecular inhibitors or monoclonal antibodies of each of these signaling pathways have been studied in clinical trials. This review focuses on the involvement of Hey proteins in malignant carcinoma progression and provides valuable therapeutic information for anticancer treatment. Mol Cancer Ther; 16(5); 775–86. ©2017 AACR.

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Dual Targeting of Epithelial Ovarian Cancer Via Folate Receptor {alpha} and the Proton-Coupled Folate Transporter with 6-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates

Folate uptake in epithelial ovarian cancer (EOC) involves the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT), both facilitative transporters and folate receptor (FR) α. Although in primary EOC specimens, FRα is widely expressed and increases with tumor stage, PCFT was expressed independent of tumor stage (by real-time RT-PCR and IHC). EOC cell line models, including cisplatin sensitive (IGROV1 and A2780) and resistant (SKOV3 and TOV112D) cells, expressed a 17-fold range of FRα and similar amounts (within ~2-fold) of PCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates AGF94 and AGF154 exhibited potent antiproliferative activities toward all of the EOC cell lines, reflecting selective cellular uptake by FRα and/or PCFT over RFC. When IGROV1 cells were pretreated with AGF94 at pH 6.8, clonogenicity was potently inhibited, confirming cell killing. FRα was knocked down in IGROV1 cells with lentiviral shRNAs. Two FRα knockdown clones (KD-4 and KD-10) showed markedly reduced binding and uptake of [³H]folic acid and [³H]AGF154 by FRα, but maintained high levels of [³H]AGF154 uptake by PCFT compared to nontargeted control cells. In proliferation assays, KD-4 and KD-10 cells preserved in vitro inhibition by AGF94 and AGF154, compared to a nontargeted control, attributable to residual FRα- and substantial PCFT-mediated uptake. KD-10 tumor xenografts in severe-compromised immune-deficient mice were likewise sensitive to AGF94. Collectively, our results demonstrate the substantial therapeutic potential of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with dual targeting of PCFT and FRα toward EOCs that express a range of FRα, along with PCFT, as well as cisplatin resistance. Mol Cancer Ther; 16(5); 819–30. ©2017 AACR.

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Nodal Signaling as a Developmental Therapeutics Target in Oncology

The tumor microenvironment is a vital feature of oncogenesis and tumor progression. There are several parallels between cancer cells and early developmental stem cells, including their plasticity and signaling mechanisms. In early fetal development, Nodal is expressed for endodermal and mesodermal differentiation. This expression has been shown reemerge in the setting of epithelial cancers, such as breast and melanoma. High Nodal expression correlates to an aggressive tumor grade in these malignancies. Nodal signal begins with its interaction with its coreceptor, Cripto-1, leading to activation of Smad2/Smad3 and ultimately downstream transcription and translation. Lefty is the natural inhibitor of Nodal and controls Nodal signaling during fetal development. However, cancer cells lack the presence of Lefty, thus leading to uncontrolled tumor growth. Given this understanding, inhibition of the Nodal pathway offers a new novel therapeutic target in oncology. Mol Cancer Ther; 16(5); 787–92. ©2017 AACR.

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Characterization of the Anti-PD-1 Antibody REGN2810 and Its Antitumor Activity in Human PD-1 Knock-In Mice

The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti–PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1–dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861–70. ©2017 AACR.

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Acquired Resistance to the Hsp90 Inhibitor, Ganetespib, in KRAS-Mutant NSCLC Is Mediated via Reactivation of the ERK-p90RSK-mTOR Signaling Network

Approximately 25% of non–small cell lung cancer (NSCLC) patients have KRAS mutations, and no effective therapeutic strategy exists for these patients. The use of Hsp90 inhibitors in KRAS-mutant NSCLC appeared to be a promising approach, as these inhibitors target many KRAS downstream effectors; however, limited clinical efficacy has been observed due to resistance. Here, we examined the mechanism(s) of acquired resistance to the Hsp90 inhibitor, ganetespib, and identified novel and rationally devised Hsp90 inhibitor combinations, which may prevent and overcome resistance to Hsp90 inhibitors. We derived KRAS-mutant NSCLC ganetespib-resistant cell lines to identify the resistance mechanism(s) and identified hyperactivation of RAF/MEK/ERK/RSK and PI3K/AKT/mTOR pathways as key resistance mechanisms. Furthermore, we found that ganetespib-resistant cells are "addicted" to these pathways, as ganetespib resistance leads to synthetic lethality to a dual PI3K/mTOR, a PI3K, or an ERK inhibitor. Interestingly, the levels and activity of a key activator of the mTOR pathway and an ERK downstream target, p90 ribosomal S6 kinase (RSK), were also increased in the ganetespib-resistant cells. Genetic or pharmacologic inhibition of p90RSK in ganetespib-resistant cells restored sensitivity to ganetespib, whereas p90RSK overexpression induced ganetespib resistance in naïve cells, validating p90RSK as a mediator of resistance and a novel therapeutic target. Our studies offer a way forward for Hsp90 inhibitors through the rational design of Hsp90 inhibitor combinations that may prevent and/or overcome resistance to Hsp90 inhibitors, providing an effective therapeutic strategy for KRAS-mutant NSCLC. Mol Cancer Ther; 16(5); 793–804. ©2017 AACR.

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Fluorinated N,N'-Diarylureas As Novel Therapeutic Agents Against Cancer Stem Cells

Colorectal cancer is the second-leading cause of cancer-related mortality in the United States. More than 50% of patients with colorectal cancer will develop local recurrence or distant organ metastasis. Cancer stem cells play a major role in the survival and metastasis of cancer cells. In this study, we examined the effects of novel AMP-activated protein kinase (AMPK) activating compounds on colorectal cancer metastatic and stem cell lines as potential candidates for chemotherapy. We found that activation of AMPK by all fluorinated N,N-diarylureas (FND) compounds at micromolar levels significantly inhibited the cell-cycle progression and subsequent cellular proliferation. In addition, we demonstrated that select FNDs significantly increased apoptosis in colorectal cancer metastatic and cancer stem cells. Therefore, FNDs hold considerable promise in the treatment of metastatic colorectal cancer, through elimination of both regular cancer cells and cancer stem cells. Mol Cancer Ther; 16(5); 831–7. ©2017 AACR.

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Characterization of a Dual Rac/Cdc42 Inhibitor MBQ-167 in Metastatic Cancer

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell-cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC₅₀, 1,100 nmol/L) inhibits cancer cell migration and viability and reduces tumor growth, metastasis, and angiogenesis in vivo. Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC₅₀ values of 103 and 78 nmol/L, respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of STAT3, without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial-to-mesenchymal transition by a loss of cell polarity and inhibition of cell surface actin-based extensions to ultimately result in detachment from the substratum. Prolonged incubation (120 hours) in MBQ-167 decreases metastatic cancer cell viability with a GI₅₀ of approximately 130 nmol/L, without affecting noncancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167–mediated G₂–M cell-cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by approximately 90%. In conclusion, MBQ-167 is 10x more potent than other currently available Rac/Cdc42 inhibitors and has the potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42. Mol Cancer Ther; 16(5); 805–18. ©2017 AACR.

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Selective Killing of SMARCA2- and SMARCA4-deficient Small Cell Carcinoma of the Ovary, Hypercalcemic Type Cells by Inhibition of EZH2: In Vitro and In Vivo Preclinical Models

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

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Modulating Therapeutic Activity and Toxicity of Pyrrolobenzodiazepine Antibody-Drug Conjugates with Self-Immolative Disulfide Linkers

A novel disulfide linker was designed to enable a direct connection between cytotoxic pyrrolobenzodiazepine (PBD) drugs and the cysteine on a targeting antibody for use in antibody–drug conjugates (ADCs). ADCs composed of a cysteine-engineered antibody were armed with a PBD using a self-immolative disulfide linker. Both the chemical linker and the antibody site were optimized for this new bioconjugation strategy to provide a highly stable and efficacious ADC. This novel disulfide ADC was compared with a conjugate containing the same PBD drug, but attached to the antibody via a peptide linker. Both ADCs had similar efficacy in mice bearing human tumor xenografts. Safety studies in rats revealed that the disulfide-linked ADC had a higher MTD than the peptide-linked ADC. Overall, these data suggest that the novel self-immolative disulfide linker represents a valuable way to construct ADCs with equivalent efficacy and improved safety. Mol Cancer Ther; 16(5); 871–8. ©2017 AACR.

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Preclinical Antitumor Efficacy of BAY 1129980--a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non–small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody–drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893–904. ©2017 AACR.

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Inhibition of Isoprenylcysteine Carboxylmethyltransferase Induces Cell-Cycle Arrest and Apoptosis through p21 and p21-Regulated BNIP3 Induction in Pancreatic Cancer

Pancreatic cancer remains one of the most difficult to treat human cancers despite recent advances in targeted therapy. Inhibition of isoprenylcysteine carboxylmethyltransferase (ICMT), an enzyme that posttranslationally modifies a group of proteins including several small GTPases, suppresses proliferation of some human cancer cells. However, the efficacy of ICMT inhibition on human pancreatic cancer has not been evaluated. In this study, we have evaluated a panel of human pancreatic cancer cell lines and identified those that are sensitive to ICMT inhibition. In these cells, ICMT suppression inhibited proliferation and induced apoptosis. This responsiveness to ICMT inhibition was confirmed in in vivo xenograft tumor mouse models using both a small-molecule inhibitor and shRNA-targeting ICMT. Mechanistically, we found that, in sensitive pancreatic cancer cells, ICMT inhibition induced mitochondrial respiratory deficiency and cellular energy depletion, leading to significant upregulation of p21. Furthermore, we characterized the role of p21 as a regulator and coordinator of cell signaling that responds to cell energy depletion. Apoptosis, but not autophagy, that is induced via p21-activated BNIP3 expression accounts for the efficacy of ICMT inhibition in sensitive pancreatic cancer cells in both in vitro and in vivo models. In contrast, cells resistant to ICMT inhibition demonstrated no mitochondria dysfunction or p21 signaling changes under ICMT suppression. These findings not only identify pancreatic cancers as potential therapeutic targets for ICMT suppression but also provide an avenue for identifying those subtypes that would be most responsive to agents targeting this critical enzyme. Mol Cancer Ther; 16(5); 914–23. ©2017 AACR.

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Highly Potent, Anthracycline-based Antibody-Drug Conjugates Generated by Enzymatic, Site-specific Conjugation

Antibody–drug conjugates (ADC) are highly potent and specific antitumor drugs, combining the specific targeting of mAbs with the potency of small-molecule toxic payloads. ADCs generated by conventional chemical conjugation yield heterogeneous mixtures with variable pharmacokinetics, stability, safety, and efficacy profiles. To address these issues, numerous site-specific conjugation technologies are currently being developed allowing the manufacturing of homogeneous ADCs with predetermined drug-to-antibody ratios. Here, we used sortase-mediated antibody conjugation (SMAC) technology to generate homogeneous ADCs based on a derivative of the highly potent anthracycline toxin PNU-159682 and a noncleavable peptide linker, using the anti-HER2 antibody trastuzumab (part of Kadcyla) and the anti-CD30 antibody cAC10 (part of Adcetris). Characterization of the resulting ADCs in vitro and in vivo showed that they were highly stable and exhibited potencies exceeding those of ADCs based on conventional tubulin-targeting payloads, such as Kadcyla and Adcetris. The data presented here suggest that such novel and highly potent ADC formats may help to increase the number of targets available to ADC approaches, by reducing the threshold levels of target expression required. Mol Cancer Ther; 16(5); 879–92. ©2017 AACR.

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Lipid Nanoparticle-Mediated Delivery of Anti-miR-17 Family Oligonucleotide Suppresses Hepatocellular Carcinoma Growth

Hepatocellular carcinoma (HCC) is one of the most common human malignancies with poor prognosis and urgent unmet medical need. Aberrant expression of multiple members of the miR-17 family are frequently observed in HCC, and their overexpression promotes tumorigenic properties of HCC cells. However, whether pharmacologic inhibition of the miR-17 family inhibits HCC growth remains unknown. In this study, we validated that the miR-17 family was upregulated in a subset of HCC tumors and cell lines and its inhibition by a tough decoy inhibitor suppressed the growth of Hep3B and HepG2 cells, which overexpress the miR-17 family. Furthermore, inhibition of the miR-17 family led to a global derepression of direct targets of the family in all three HCC cell lines tested. Pathway analysis of the deregulated genes indicated that the genes associated with TGFβ signaling pathway were highly enriched in Hep3B and HepG2 cells. A miR-17 family target gene signature was established and used to identify RL01-17(5), a lipid nanoparticle encapsulating a potent anti-miR-17 family oligonucleotide. To address whether pharmacologic modulation of the miR-17 family can inhibit HCC growth, RL01-17(5) was systemically administrated to orthotopic Hep3B xenografts. Suppression of Hep3B tumor growth in vivo was observed and tumor growth inhibition correlated with induction of miR-17 family target genes. Together, this study provides proof-of-concept for targeting the miR-17 family in HCC therapy. Mol Cancer Ther; 16(5); 905–13. ©2017 AACR.

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Role of STAT3 and FOXO1 in the Divergent Therapeutic Responses of Non-metastatic and Metastatic Bladder Cancer Cells to miR-145

Although miR-145 is the most frequently downregulated miRNA in bladder cancer, its exact stage association and downstream effector have not been defined. Here, we found that miR-145 was upregulated in human patients with bladder cancer with lymph node metastasis and in metastatic T24T cell line. Forced expression of miR-145 promoted anchorage-independent growth of T24T cells accompanied by the downregulation of forkhead box class O1 (FOXO1). In contrast, in non-metastatic T24 cells, miR-145 overexpression inhibited cell growth with upregulation of FOXO1, and the knockdown of FOXO1 abolished the miR-145–mediated inhibition of cell growth. Mechanistic studies revealed that miR-145 directly bound to and attenuated 3'-untranslated region (UTR) activity of foxo1 mRNA in both T24 and T24T cells. Interestingly, miR-145 suppressed STAT3 phosphorylation at Tyr705 and increased foxo1 promoter transcriptional activity in T24 cells, but not in T24T cells, suggesting a role of STAT3 in the divergent responses to miR-145. Supporting this was our finding that STAT3 knockdown mimicked miR-145–mediated upregulation of FOXO1 in T24T cells and inhibition of anchorage-independent growth. Consistently, ectopic expression of miR-145 promoted tumor formation of xenograft T24T cells, whereas such promoting effect became inhibitory due to specific knockdown of STAT3. Together, our findings demonstrate the stage-specific association and function of miR-145 in bladder cancers and provide novel insights into the therapeutic targeting of miR-145. Mol Cancer Ther; 16(5); 924–35. ©2017 AACR.

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Calcium-Dependent Enhancement by Extracellular Acidity of the Cytotoxicity of Mitochondrial Inhibitors against Melanoma

Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Because acidity potentiates malignant phenotypes, therapeutic strategies that enhance the targeting of oncogenic mechanisms in an acidic microenvironment should be effective. We report here that drugs which abrogate mitochondrial respiration show enhanced cytotoxicity against melanoma cells in a normoxic but acidic extracellular pH, independent from P53 mutations, BRAF (V600E) mutations, and/or resistance against BRAF inhibitors. Conversely, the cytotoxicity against melanoma cells of mitochondrial inhibitors is impaired by a neutral or alkaline extracellular pH, and in vivo systemic alkalinization with NaHCO₃ enhanced subcutaneous tumor growth and lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin. Intracellular calcium (Ca²⁺) was significantly increased in melanoma cells treated with mitochondrial inhibitors at an acidic extracellular pH and an intracellular Ca²⁺ chelator, BAPTA/AM, inhibited cytoplasmic Ca²⁺ as well as melanoma cell death. Surprisingly, ROS scavengers synergized with increased apoptosis in cells treated with mitochondrial inhibitors, suggesting that ROS contributes to cell survival in this context. Notably, the cytotoxic enhancement of mitochondrial inhibitors by acidity was distinct from PGC1alpha-driven mitochondrial addiction, from therapy-induced senescence, and from slow, JARID1B-high–associated cell cycling, all of which have been shown to promote vulnerability to mitochondrial inhibition. These data indicate that extracellular pH profoundly modulates the cytotoxicity of mitochondrial inhibitors against cancer cells. Mol Cancer Ther; 16(5); 936–47. ©2017 AACR.

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Cell-Free DNA from Ascites and Pleural Effusions: Molecular Insights into Genomic Aberrations and Disease Biology

Collection of cell-free DNA (cfDNA) from the blood of individuals with cancer has permitted noninvasive tumor genome analysis. Detection and characterization of cfDNA in ascites and pleural effusions have not yet been reported. Herein, we analyzed cfDNA in the ascites and pleural effusions from six individuals with metastatic cancer. In all cases, cfDNA copy number variations (CNV) were discovered within the effusate. One individual had a relevant alteration with a high copy amplification in EGFR in a never smoker with lung cancer, who showed only MDM2 and CDK4 amplification in a prior tissue biopsy. Another subject with metastatic breast cancer had cytology-positive ascites and an activating PIK3CA mutation identified in the tissue, blood, and ascites collectively. This individual had tumor regression after the administration of the mTOR inhibitor everolimus and had evidence of chromotripsis from chromosomal rearrangements noted in the cell-free ascitic fluid. These results indicate that cfDNA from ascites and pleural effusions may provide additional information not detected with tumor and plasma cell-free DNA molecular characterization, and a context for important insights into tumor biology and clonal dynamic change within primary tumor and metastatic deposits. Mol Cancer Ther; 16(5); 948–55. ©2017 AACR.

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