Involvement of the AKT Pathway in Resistance to Erlotinib and Cabozantinib in Triple-Negative Breast Cancer Cell Lines.

Resistance to protein tyrosine kinase inhibitors (TKIs) presents a significant challenge in therapeutic target development for cancers such as triple-negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that adaptive responses in regulatory kinase networks within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. We observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion, and activation of EGFR or c-Met downstream signaling (respectively). Using a SILAC (Stable Isotope Labeling of Amino acids in Cell Culture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib or cabozantinib resistance on the phosphoproteome, proteome, and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozantinib resistance and confirmed the contribution of AKT1 to erlotinib resistance in TNBC-resistant cell lines.

Reduced Zeb1 Expression in Prostate Cancer Cells Leads to an Aggressive Partial-EMT Phenotype Associated with Altered Global Methylation Patterns.

Prostate cancer is the most common cancer in American men and the second leading cause of cancer-related death. Most of these deaths are associated with metastasis, a process involving the epithelial-to-mesenchymal (EMT) transition. Furthermore, growing evidence suggests that partial-EMT (p-EMT) may lead to more aggressive disease than complete EMT. In this study, the EMT-inducing transcription factor Zeb1 was knocked down in mesenchymal PC-3 prostate cancer cells (Zeb1KD) and resulting changes in cellular phenotype were assessed using protein and RNA analysis, invasion and migration assays, cell morphology assays, and DNA methylation chip analysis. Inducible knockdown of Zeb1 resulted in a p-EMT phenotype including co-expression of epithelial and mesenchymal markers, a mixed epithelial/mesenchymal morphology, increased invasion and migration, and enhanced expression of p-EMT markers relative to PC-3 mesenchymal controls (p ≤ 0.05). Treatment of Zeb1KD cells with the global de-methylating drug 5-azacytidine (5-aza) mitigated the observed aggressive p-EMT phenotype (p ≤ 0.05). DNA methylation chip analysis revealed 10 potential targets for identifying and/or targeting aggressive p-EMT prostate cancer in the future. These findings provide a framework to enhance prognostic and/or therapeutic options for aggressive prostate cancer in the future by identifying new p-EMT biomarkers to classify patients with aggressive disease who may benefit from 5-aza treatment.

Everolimus in Advanced Breast Cancer: A Systematic Review and Meta-analysis.

BACKGROUND: Everolimus plus exemestane is approved for the treatment of hormone receptor-positive metastatic breast cancer (MBC) after progression on nonsteroidal aromatase inhibitors. The role of everolimus is less well defined in other breast cancer phenotypes and in combination with other drugs. OBJECTIVES: We conducted a systematic review and meta-analysis to assess the efficacy and safety of adding everolimus to standard of care (SoC) in MBC regardless of tumor phenotype and treatment type. METHODS: The electronic databases PubMed and EMBASE were searched for eligible randomized trials. Pooled hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS) and pooled risk ratios (RR) and odds ratios for objective response rates, disease control rate (DCR), and grade 3 or higher toxicity were meta-analyzed. Subgroup analyses compared survival outcomes by tumor phenotype. RESULTS: Data from 2826 patients from eight trials were analyzed. The addition of everolimus to SoC reduced the risk of disease progression by 29% (HR 0.71; 95% confidence interval [CI] 0.56-0.90). This did not translate into an OS benefit (HR 0.95; 95% CI 0.80-1.13). In addition, everolimus improved the DCR (RR 0.82; 95% CI 0.68-0.98), whereas it increased the risk of developing grade 3 or higher toxicity. The PFS benefit was more prominent for patients with hormone receptor-positive (+)/human epidermal growth factor receptor 2 (HER2)-negative (-) disease. For the HER2 (+) subgroup, the PFS benefit was restricted to patients with hormone receptor (-) disease. CONCLUSIONS: Everolimus reduces the risk of disease progression in hormone receptor (+) MBC. In patients with HER2 (+) disease, the benefit is limited for those with hormone receptor (-) disease. Given the approval and use of newer drugs in MBC, clinical trials and real-world data are needed to confirm the benefit of everolimus and define the best treatment sequence strategy to adopt in that setting.

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

Overview of the Canadian Clinician Investigator Trainees' research presented at the 2019 CSCI-CITAC Joint Meeting.

The 2019 Annual General Meeting and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de Recherche Clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada / Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was held in Banff, Alberta on November 8-10th, 2019. The theme was "Positioning Early Career Investigators for Success: Strategy and Resilience". Lectures and workshops provided knowledge and tools to facilitate the attendees' development as clinician investigators. Dr. Jason Berman (President of CSCI/SCRC), Elina Cook (President of CITAC/ACCFC) and Drs. Doreen Rabi and Zelma Kiss (University of Calgary Organizing Co-Chairs) gave opening presentations. The keynote speakers were Dr. William Foulkes (McGill University) (Distinguished Scientist Award winner) and Dr. Andrés Finzi (Université de Montréal) (Joe Doupe Young Investigator Award winner). Dr. Robert Bortolussi (Dalhousie University) received the Distinguished Service Award for his work as the Editor-in-Chief of Clinical and Investigative Medicine and for being instrumental in the development of the Canadian Child Health Clinician Scientist Program. This meeting was the first to host a panel discussion with Drs. Stephen Robbins and Marcello Tonelli from the Canadian Institutes of Health Research. Workshops on communication, career planning and work-life balance were hosted by André Picard and Drs. Todd Anderson, Karen Tang, William Ghali, May Lynn Quan, Alicia Polachek and Shannon Ruzycki. The AGM showcased 90 presentations from clinician investigator trainees from across Canada. Most of the abstracts are summarized in this review. Eight outstanding abstracts were selected for oral presentation at the President's Forum.

Apple derived cellulose scaffolds for 3D mammalian cell culture.

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.