Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

Abstract

Background

During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance.

Methods

The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay.

Results

Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2.

Conclusion

Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.

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PD-L1 Expression in Melanoma: A Quantitative Immunohistochemical Antibody Comparison

Purpose: PD-L1 expression in the pretreatment tumor microenvironment enriches for response to anti-PD-1/PD-L1 therapies. The purpose of this study was to quantitatively compare the performance of five monoclonal anti-PD-L1 antibodies used in recent landmark publications.

Experimental Design: PD-L1 IHC was performed on 34 formalin-fixed paraffin-embedded archival melanoma samples using the 5H1, SP142, 28-8, 22C3, and SP263 clones. The percentage of total cells (including melanocytes and immune cells) demonstrating cell surface PD-L1 staining, as well as intensity measurements/H-scores, were assessed for each melanoma specimen using a computer-assisted platform. Staining properties were compared between antibodies.

Results: Strong correlations were observed between the percentage of PD-L1(+) cells across all clones studied (R² = 0.81–0.96). When present, discordant results were attributable to geographic heterogeneity of the melanoma tissue section rather than differences in PD-L1 antibody staining characteristics. PD-L1 intensity/H-scores strongly correlated with percentage of PD-L1(+) cells (R² > 0.78, all clones).

Conclusions: The 5H1, SP142, 28-8, 22C3, and SP263 clones all demonstrated similar performance characteristics when used in a standardized IHC assay on melanoma specimens. Reported differences in PD-L1 IHC assays using these antibodies are thus most likely due to assay characteristics beyond the antibody itself. Our findings also argue against the inclusion of an intensity/H-score in chromogenic PD-L1 IHC assays. Clin Cancer Res; 23(16); 4938–44. ©2017 AACR.

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A Gene Signature for Selecting Benefit from Hypoxia Modification of Radiotherapy for High-Risk Bladder Cancer Patients

Purpose: Hypoxia modification improves overall survival in muscle-invasive bladder cancer patients who undergo radiotherapy. There is evidence that hypoxic tumors benefit most from hypoxia modification. The study aimed to identify or derive a hypoxia gene signature that predicts benefit from hypoxia-modifying treatment in bladder cancer.

Experimental Design: Published hypoxia signatures were tested and a new one derived by analyzing bladder cancer transcriptomic data from public databases. Tumor samples were available from the BCON phase III randomized trial of radiotherapy alone or with carbogen and nicotinamide (CON). Gene expression data were generated for 151 tumors using Affymetrix Human 1.0 Exon ST arrays and used for independent validation.

Results: A 24-gene signature was derived, which was prognostic in four of six independent surgical cohorts (n = 679; meta HR, 2.32; 95% CI, 1.73–3.12; P < 0.0001). The signature was also prognostic in BCON patients receiving radiotherapy alone (n = 75; HR for local relapse-free survival, 2.37; 95% CI, 1.26–4.47; P = 0.0076). The signature predicted benefit from CON (n = 76; HR, 0.47; 95% CI, 0.26–0.86; P = 0.015). Prognostic significance (P = 0.017) and predictive significance (P = 0.058) remained after adjusting for clinicopathologic variables. A test for interaction between hypoxia status and treatment arms was significant (P = 0.0094).

Conclusions: A 24-gene hypoxia signature has strong and independent prognostic and predictive value for muscle-invasive bladder cancer patients. The signature can aid identification of patients likely to benefit from the addition of carbogen and nicotinamide to radiotherapy. Clin Cancer Res; 23(16); 4761–8. ©2017 AACR.

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Venetoclax in Patients with Previously Treated Chronic Lymphocytic Leukemia

Venetoclax is the first BCL2 inhibitor to enter routine clinical practice. It is an orally bioavailable small molecule that binds BCL2 very specifically. Acting as a pharmacologic mimic of the proteins that initiate apoptosis (a so-called BH3 mimetic), venetoclax rapidly induces apoptosis in chronic lymphocytic leukemia (CLL) cells, which express high levels of BCL2 and rely on it to maintain their survival. As a single agent, daily venetoclax treatment induced durable responses in 79% of patients with relapsed or refractory CLL or small lymphocytic lymphoma in a phase I study, including complete remissions in 20% of patients. Its use was approved by the FDA in April 2016 for patients with previously treated del(17p) CLL on the basis of a single-arm phase II trial demonstrating a 79% response rate and an estimated 1-year progression-free survival of 72% with 400 mg/day continuous therapy. This review focuses on venetoclax, its mechanism of action, pharmacology, and clinical trial data and seeks to place it in the context of rapid advances in therapy for patients with relapsed CLL, especially those with del(17p) CLL. Clin Cancer Res; 23(16); 4527–33. ©2017 AACR.

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Genomic Analysis of Thymic Epithelial Tumors Identifies Novel Subtypes Associated with Distinct Clinical Features

Purpose: To reconcile the heterogeneity of thymic epithelial tumors (TET) and gain deeper understanding of the molecular determinants of TETs, we set out to establish a clinically relevant molecular classification system for these tumors.

Experimental Design: Molecular subgrouping of TETs was performed in 120 patients from The Cancer Genome Atlas using a multidimensional approach incorporating analyses of DNA mutations, mRNA expression, and somatic copy number alterations (SCNA), and validated in two independent cohorts.

Results: Four distinct molecular subtypes of TETs were identified. The most commonly identified gene mutation was a missense mutation in General Transcription Factor II-I (GTF2I group), which was present in 38% of patients. The next group was identified by unsupervised mRNA clustering of GTF2I wild-type tumors and represented TETs enriched in expression of genes associated with T-cell signaling (TS group; 33%). The remaining two groups were distinguished by their degree of chromosomal stability (CS group; 8%) or instability (CIN group; 21%) based upon SCNA analyses. Disease-free survival and overall survival were favorable in the GTF2I group and unfavorable in the CIN group. These molecular subgroups were associated with TET histology and clinical features including disease-free survival. Finally, we demonstrate high expression of PD1 mRNA and correlation of PD1 and CD8A in the TS subgroup.

Conclusions: Molecular subtyping of TETs is associated with disease-free and overall survival. Classification of TETs by a molecular framework could aid in the refinement of staging and in the discovery and development of rational treatment options for patients with TETs. Clin Cancer Res; 23(16); 4855–64. ©2017 AACR.

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U.S. Food and Drug Administration Approval Summary: Atezolizumab for Metastatic Non-Small Cell Lung Cancer

On October 18, 2016, the FDA approved atezolizumab (TECENTRIQ; Genentech, Inc.) for treatment of patients with metastatic non–small cell lung cancer (mNSCLC) whose disease progressed during or following platinum-containing chemotherapy. Approval was based on demonstration of clinically meaningful improvements in overall survival (OS) and an acceptable safety profile in two randomized clinical trials (OAK and POPLAR). Median OS in OAK, a phase III trial, was 13.8 months [95% confidence interval (CI), 11.8–15.7] in the atezolizumab arm compared with 9.6 months (95% CI, 8.6–11.2) in the docetaxel arm [hazard ratio (HR) = 0.74; 95% CI, 0.63–0.87; P = 0.0004]. Median OS in POPLAR, a phase II trial, was 12.6 months (95% CI, 9.7–16.0) and 9.7 months (95% CI, 8.6–12.0; HR = 0.69; 95% CI, 0.52–0.92) for the atezolizumab and docetaxel arms, respectively. In patients treated with atezolizumab, the most common (≥20%) adverse reactions were fatigue, decreased appetite, dyspnea, cough, nausea, musculoskeletal pain, and constipation; the most common (≥2%) grade 3 to 4 adverse events were dyspnea, pneumonia, hypoxia, hyponatremia, fatigue, anemia, musculoskeletal pain, aspartate aminotransferase increase, alanine aminotransferase increase, dysphagia, and arthralgia. Clinically significant immune-related adverse events for patients receiving atezolizumab included 1.4% incidence each of grade 3 to 4 pneumonitis, hepatitis, colitis, and thyroid disease. Clin Cancer Res; 23(16); 4534–9. ©2017 AACR.

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Molecular Pathways: Targeting the Protein Kinase Wee1 in Cancer

Wee1 is a protein kinase that regulates the G₂ checkpoint and prevents entry into mitosis in response to DNA damage. Cyclin-dependent kinases (CDK) are a family of 14 serine/threonine protein kinases that coordinate the progression through the cell cycle. The Cdc2/cyclin B complex controls the progression from G₂ into mitosis. There are two mechanisms by which the G₂ checkpoint is initiated in response to DNA damage: phosphorylation of Cdc25c by CHK1 and of the Wee1 kinase, which phosphorylates Cdc2. Blockade at the G₂ checkpoint is especially important for p53-mutant cells because these tumors mainly rely on DNA repair at the G₂ checkpoint. AZD1775 (formerly MK-1775) is a small-molecule, pyrazol-pyrimidine derivative and potent and ATP-competitive specific inhibitor of the Wee1 kinase. Several preclinical and clinical studies demonstrated encouraging antitumor effects with manageable side effects of the combination of Wee1 inhibition and DNA-damaging agents. Promising combination schedules are being investigated at the moment, for example, combining PARP inhibition and Wee1 inhibition. Also, a weekly schedule with carboplatin and AZD1775 warrants investigation aimed at further improving the antitumor effect. Clin Cancer Res; 23(16); 4540–4. ©2017 AACR.

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