Moumita Chatterjee

Caveolin-1 is Associated with Tumor Progression and Confers a Multi-Modality Resistance Phenotype in Pancreatic Cancer

Caveolin-1 (Cav-1) is a 21 kDa protein enriched in caveolae, and has been implicated in oncogenic cell transformation, tumorigenesis, and metastasis. We explored roles for Cav-1 in pancreatic cancer (PC) prognostication, tumor progression, resistance to therapy, and whether targeted downregulation could lead to therapeutic sensitization. Cav-1 expression was assessed in cell lines, mouse models, and patient samples, and knocked down in order to compare changes in proliferation, invasion, migration, response to chemotherapy and radiation, and tumor growth. We found Cav-1 is overexpressed in human PC cell lines, mouse models, and human pancreatic tumors, and is associated with worse tumor grade and clinical outcomes. In PC cell lines, disruption/depletion of caveolae/Cav-1 reduces proliferation, colony formation, and invasion. Radiation and chemotherapy up-regulate Cav-1 expression, while Cav-1 depletion induces both chemosensitization and radiosensitization through altered apoptotic and DNA repair signaling. In vivo, Cav-1 depletion significantly attenuates tumor initiation and growth. Finally, Cav-1 depletion leads to altered JAK/STAT, JNK, and Src signaling in PC cells. Together, higher Cav-1 expression is correlated with worse outcomes, is essential for tumor growth and invasion (both in vitro and in vivo), is responsible for promoting resistance to therapies, and may serve as a prognostic/predictive biomarker and target in PC.

Stromal Caveolin-1 Is Associated With Response and Survival in a Phase II Trial of nab-Paclitaxel With Carboplatin for Advanced NSCLC Patients

UNLABELLED In this phase II trial, carboplatin with nanoparticle albumin-bound (nab)-paclitaxel as first-line therapy for advanced non-small-cell lung cancer (NSCLC) was evaluated. Most patients had squamous cell histology. Tumor-associated stromal caveolin-1 (Cav-1) expression was correlated with improved response rate and survival in NSCLC patients who received nab-paclitaxel in this phase II trial. These results suggest Cav-1 might serve as a potential biomarker in this patient population. BACKGROUND The combination of bevacizumab with platinum-based chemotherapy results in greater response rate (RR) and overall survival (OS) in advanced non-small-cell lung cancer (NSCLC). Bevacizumab is contraindicated in patients with squamous histology or hemoptysis. Nanoparticle albumin-bound (nab)-paclitaxel is a novel formulation of paclitaxel with greater dose tolerance and improved efficacy. We hypothesized that nab-paclitaxel and carboplatin would be superior to alternative doublets in advanced NSCLC patients ineligible for bevacizumab. PATIENTS AND METHODS We conducted a single-arm phase II trial (NCT00729612) with carboplatin and nab-paclitaxel on day 1 of a 21-day cycle to evaluate RR (primary end point), safety, toxicity, and OS. Eligibility included: squamous histology, hemoptysis, or ongoing anticoagulation. Correlative studies included immunohistochemistry for secreted protein acid rich in cysteine (SPARC) and caveolin-1 (Cav-1). RESULTS Sixty-three patients were enrolled. Most patients had squamous cell carcinoma (n = 48); other reasons for eligibility included hemoptysis (n = 11) and anticoagulation (n = 2). Toxicity Grade ≥ 3/4 included neuropathy, cytopenias, and fatigue. RR was 38% (24 partial response/0 complete response); 20 patients had stable disease (32%). Median progression-free survival was 5 months and median OS was 9.7 months. Immunohistochemistry for SPARC and Cav-1 was performed in 38 and 37 patients respectively. Although no association was found for SPARC expression in tumor or stroma with RR or OS, we found that higher Cav-1 levels in tumor-associated stroma was associated with improved RR and OS. CONCLUSION Carboplatin and nab-paclitaxel every 21 days demonstrated promising efficacy with tolerable toxicity in NSCLC patients ineligible for bevacizumab therapy. Further analysis and validation of Cav-1 and SPARC expression in tumor and stromal compartments as prognostic and/or predictive biomarkers of NSCLC or nab-paclitaxel treatment is warranted.

Initial testing (stage 1) of the tubulin binding agent nanoparticle albumin-bound (nab) paclitaxel (Abraxane ® ) by the Pediatric Preclinical Testing Program (PPTP)

Nanoparticle albumin‐bound paclitaxel (nab‐paclitaxel, Abraxane®) is FDA approved for the treatment of several adult cancers. Antimitotic agents are essential components for curative therapy of pediatric solid tumors, although taxanes have shown limited activity. Because of the novel formulation, nab‐paclitaxel was evaluated against a limited series of Pediatric Preclinical Testing Program (PPTP) solid tumors.

Caveolae-Mediated Endocytosis Is Critical for Albumin Cellular Uptake and Response to Albumin-Bound Chemotherapy

Nab-paclitaxel, a nanoparticle conjugate of paclitaxel to human albumin, exhibits efficacy in pancreatic cancer, non-small cell lung cancer and breast cancer. However, there is a lack of predictive biomarkers to identify patients who might benefit most from its administration. This study addresses this gap in knowledge by identifying that caveolin-1 (Cav-1) is a candidate mechanism-based biomarker. Caveolae are small membrane invaginations important for transendothelial albumin uptake. Cav-1, the principal structural component of caveolae, is overexpressed in the cancers noted above that respond to nab-paclitaxel. Thus, we hypothesized that Cav-1 may be critical for albumin uptake in tumors and perhaps determine their response to this drug. Cav-1 protein levels correlated positively with nab-paclitaxel sensitivity. RNAi-mediated attenuation of Cav-1 expression reduced uptake of albumin and nab-paclitaxel in cancer cells and rendered them resistant to nab-paclitaxel-induced apoptosis. Conversely, Cav-1 overexpression enhanced sensitivity to nab-paclitaxel. Selection for cellular resistance to nab-paclitaxel in cell culture correlated with a loss of Cav-1 expression. In mouse xenograft models, cancer cells, where Cav-1 was attenuated, exhibited resistance to the antitumor effects of nab-paclitaxel therapy. Overall, our findings suggest Cav-1 as a predictive biomarker for the response to nab-paclitaxel and other albumin-based cancer therapeutic drugs. Cancer Res; 77(21); 5925-37. ©2017 AACR.

MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways

Purpose: Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK inhibitors have shown promising anti-tumor responses in recent preclinical and clinical studies, and are currently being tested in combination with radiation in clinical trials. Here, we have evaluated the radiosensitizing potential of a novel MEK1/2 inhibitor GSK1120212 (GSK212,or trametinib) and evaluated whether MEK1/2 inhibition alters DNA repair mechanisms in multiple PC cell lines. Methods: Radiosensitization and DNA double-strand break (DSB) repair were evaluated by clonogenic assays, comet assay, nuclear foci formation (γH2AX, DNA-PK, 53BP1, BRCA1, and RAD51), and by functional GFP-reporter assays for homologous recombination (HR) and non-homologous end-joining (NHEJ). Expression and activation of DNA repair proteins were measured by immunoblotting. Results: GSK212 blocked ERK1/2 activity and radiosensitized multiple KRAS mutant PC cell lines. Prolonged pre-treatment with GSK212 for 24-48 hours was required to observe significant radiosensitization. GSK212 treatment resulted in delayed resolution of DNA damage by comet assays and persistent γH2AX nuclear foci. GSK212 treatment also resulted in altered BRCA1, RAD51, DNA-PK, and 53BP1 nuclear foci appearance and resolution after radiation. Using functional reporters, GSK212 caused repression of both HR and NHEJ repair activity. Moreover, GSK212 suppressed the expression and activation of a number of DSB repair pathway intermediates including BRCA1, DNA-PK, RAD51, RRM2, and Chk-1. Conclusion: GSK212 confers radiosensitization to KRAS-driven PC cells by suppressing major DNA-DSB repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of PC.

MicroRNA molecular profiling identifies potential signaling pathways conferring resistance to chemoradiation in locally-advanced rectal adenocarcinoma

Purpose There has been growing interest in using chemoradiation (CRT) for non-operative management of rectal cancer, and identifying patients who might benefit most from this approach is crucial. This study identified miRNAs (miRs) associated with clinical outcomes and treatment resistance by evaluating both pre- and post-CRT expression profiles. Methods Forty patients, 9 with pathologic complete response (pCR) and 31 with pathologic incomplete response (pIR) were included. MicroRNA was extracted from 40 pre-therapy tumor samples and 31 post-chemoradiation surgical samples with pathologic incomplete response (pIR). A generalized linear model was used to identify miRs associated with pCR. A linear mixed effects model was used to identify miRs differentially expressed before and after treatment. miR expression was dichotomized at the mean and clinical outcomes were evaluated using Cox proportional hazard modeling. Results Nine miRs were associated with pCR (p<0.05), but none were significant after false discovery rate correction. Among patients with pIR, 68 miRs were differentially expressed between the pre and post-CRT groups (FDR p<0.05). Ingenuity pathway analysis (IPA) demonstrated multiple signaling networks associated with pIR, including p38MAPK, TP53, AKT, IL-6, and RAS. Increased let-7b was correlated with increased distant metastasis (DM), worse relapse-free survival (RFS), and worse overall survival (OS) (p<0.05). Conclusions No miRs were significantly correlated with pCR. We identified miRs that were differentially expressed between pre- and post-CRT tumor samples, and these miRs implicated multiple signaling pathways that may confer resistance to CRT. In addition, we identified an association between increased let-7b and worse clinical outcomes (DM, DFS, OS).

Abstract 405: Caveolin-1 expression mediates response to albumin-bound paclitaxel

BACKGROUND: Albumin-bound chemotherapies such as nab-paclitaxel are approved to treat pancreatic cancer, non-small cell lung cancer (NSCLC), and breast cancer. Predictive biomarkers to select patients who may benefit most from nab-paclitaxel are lacking. Nab-paclitaxel is thought to enter cells through a caveolae-gp60 endocytic mechanism. Caveolin-1 (Cav-1) is a principal structural component of caveolae, and Cav-1 has been shown to be important for albumin uptake in endothelial cells. Cav-1 is known to be up-regulated in multiple tumor types, including pancreatic cancer, and certain subtypes of non-small cell lung cancer, and breast cancer. We hypothesize that Cav-1 expression may predict response to nab-paclitaxel therapy. METHODS: We correlated Cav-1 expression with nab-paclitaxel sensitivity in a panel of NSCLC and pancreatic cancer cell lines. We also assessed albumin uptake in tumor and non-tumor cell lines. We genetically depleted Cav-1 by shRNA in cells and performed cytotoxicity assays. In addition, we measured how Cav-1 levels affected albumin and nab-paclitaxel uptake into tumor cells by immunofluorescence, immunoblotting, and mass spectrometry. Annexin V flow cytometry analysis and immunoblotting for apoptosis pathway intermediates were also performed. Nab-paclitaxel resistant cell lines were created by culturing cells with increasing doses of nab-paclitaxel for extended periods of time. The role of Cav-1 expression in mediating response to nab-paclitaxel in vivo was assessed using xenograft models. RESULTS: H23 and MIA-PaCa2 tumor cells uptake more albumin compared to FHs74Int and HBEC3KT non-tumor cell lines. Higher Cav-1 expression in a panel of pancreatic cancer and NSCLC cell lines was correlated with lower IC50 for nab-paclitaxel. Cav-1 depletion resulted in reduced albumin and nab-paclitaxel uptake by tumor cells as measured by immunofluorescence, immunoblotting, and mass spectrometry. Loss of Cav-1 resulted in resistance to nab-paclitaxel but no change in sensitivity to free paclitaxel in vitro. Cav-1 down-regulation resulted in protection from nab-paclitaxel-induced apoptosis. Conversely, re-expression of Cav-1 in low-Cav-1 endogenously expressing cell lines AsPC-1 and HPAFII resulted in increased nab-paclitaxel uptake and sensitization through apoptosis. Furthermore, nab-paclitaxel resistant cells generated by prolonged exposure demonstrated downregulation of Cav-1 levels and reduced albumin uptake. Finally, genetic depletion of Cav-1 rendered tumor cells resistant to nab-paclitaxel in xenograft models, with concomitant reduced albumin uptake and activation of apoptosis. CONCLUSIONS: Our data suggest that Cav-1 expression and caveolae are critical determinants of response to nab-paclitaxel. This data supports further testing of Cav-1 as a potential predictive biomarker of response to nab-paclitaxel and potentially other albumin-bound chemotherapies. Citation Format: Terence M. Williams, Moumita Chatterjee, Ryan Robb, Marally Vedaie, Star Seum, Thirumoorthy Krishnan, Palanichamy Kamalakanan, Edgar Ben-Josef, Arnab Chakravarti. Caveolin-1 expression mediates response to albumin-bound paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 405.

Abstract B8: Caveolae-mediated endocytosis is critical for tumor cell response to nab-paclitaxel

INTRODUCTION: Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and is typified by poor outcomes. Gemcitabine with nab-paclitaxel is a standard chemotherapy combination which recently demonstrated superiority compared to gemcitabine alone in a randomized trial. However, a predictive biomarker to select patients who may benefit from nab-paclitaxel is currently lacking. Nab-paclitaxel is a nanoparticle composed of paclitaxel bound to human albumin. Caveolin-1 (Cav-1) is a 22kD structural component of caveolae, 50-100 nM invaginations of the cell membrane. Cav-1 has been demonstrated to be an important component of albumin uptake in endothelial cells, and Cav-1 is overexpressed in pancreatic cancer. We hypothesize that Cav-1 expression may predict response to nab-paclitaxel therapy. METHODS: We compared Cav-1 expression to nab-paclitaxel sensitivity in a panel of pancreatic and non-small cell lung cancer cell lines. We stably knocked down Cav-1 expression in H23 and MIA-PaCa2 cells and performed cytotoxicity assays. In addition, we measured albumin and nab-paclitaxel uptake into tumor cells with immunoblotting, immunofluorescence and mass spectrometry. Sensitivity to the therapy was confirmed by quantifying apoptosis via flow cytometry and immunoblotting for activation of apoptotic pathway intermediates. Nab-paclitaxel resistant cell lines were created by selecting resistant cells over a period of time. Results were confirmed in vivo in a nude mouse model. RESULTS: Cav-1 expression in a panel of pancreatic cancer and non-small cell lung cancer (NSCLC) cell lines negatively correlated with their corresponding IC50 values for nab-paclitaxel, indicating a positive correlation with sensitivity to nab-paclitaxel. Cav-1 depletion resulted in reduced albumin uptake in tumor cells, reduced intracellular paclitaxel, and protection from nab-paclitaxel both in vitro and in vivo, while overexpression of Cav-1 enhanced sensitivity to nab-paclitaxel. Creation of nab-paclitaxel resistant cell lines demonstrated loss of Cav-1 expression in resistant cells. In support of our preclinical data, higher Cav-1 expression in the tumor microenvironment in patients with metastatic cancer treated with nab-paclitaxel also correlates with improved response and longer survival. CONCLUSIONS: Our data indicate that Cav-1 expression mediates entry of albumin and nab-paclitaxel, subsequent response to nab-paclitaxel, and that downregulation or re-expression of Cav-1 impairs or facilitates nab-paclitaxel uptake and response, respectively. This data supports further testing of Cav-1 as a potential predictive biomarker of response to nab-paclitaxel. Citation Format: Moumita Chatterjee, Marall Vedaie, Ryan Robb, Star Seum, Adriana Estrada-Bernal, Thirumoorthy Krishnan, Palanichamy Kamalakanan, Chen Lin, Arnab Chakravarti, Terence M. Williams. Caveolae-mediated endocytosis is critical for tumor cell response to nab-paclitaxel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B8.

Abstract C31: Vemurafenib selectively radiosensitizes BRAF V600E mutant papillary and anaplastic thyroid carcinoma cells in vitro

Background: BRAF mutations are oncogenic drivers which occur in about 60% of papillary thyroid cancer (PTC) as well as a significant proportion of anaplastic thyroid cancer (ATC). BRAF mutations drive MAPK signaling and treatment resistance in thyroid cancer. BRAFV600E is the most common of these mutations in PTC, and can be selectively inhibited by vemurafenib. Methods: We used a panel of PTC and ATC cell lines to assess how the presence/absence of BRAFV600E impacts radiation sensitivity. We used radiation clonogenics, comet assays, nuclear foci formation, and western blots to determine the effect of vemurafenib on PTC and ATC cell lines. Results: Analysis of radiation clonogenics revealed relative radioresistance in cell lines containing the BRAFV600E mutation versus wild-type. Vemurafenib inhibited MAPK signaling in V600E mutants, but showed no effect on BRAF wild-type cell lines. Vemurafenib pretreatment selectively radiosensitized BRAFV600E mutants, as assessed by radiation clonogenic assays. Neutral comet assays also showed that vemurafenib impairs DNA repair in BRAFV600E lines. Furthermore, γ-H2A.x westerns and nuclear foci staining indicated that vemurafenib pretreatment decreases the ability of cells to repair double-strand DNA breaks in cell lines containing the BRAFV600E mutation. Vemurafenib also appeared to alter the kinetics of nuclear foci formation and resolution of 53BP1 as well as Rad51 in these cell lines. Conclusions: From our initial results, BRAF mutations appear to be associated with radioresistance in PTC and ATC cell lines. Vemurafenib selectively radiosensitizes both PTC and ATC cells through inhibition of DNA repair mechanisms. Together, these data suggest that combining vemurafenib and radiation may improve therapeutic control for BRAFV600E mutant thyroid cancers. Citation Format: Ryan N. Robb, Linlin Yang, Moumita Chatterjee, Moto Saji, Matt Ringel, Arnab Chakravarti, Terence Williams. Vemurafenib selectively radiosensitizes BRAF V600E mutant papillary and anaplastic thyroid carcinoma cells in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C31.

Abstract 4702: Caveolin-1 confers a multi-modality therapy resistance phenotype in pancreatic cancer cells

Caveolin-1 (Cav-1) is a 21kDa protein found in 50-100nm omega-shaped invaginations of the plasma membrane known as caveolae. It has been implicated in the processes of oncogenic cell transformation, tumorigenesis and metastasis. Cav-1 is upregulated in pancreatic cancer and has been shown to be correlated with poor prognosis. We hypothesized that Cav-1 promotes resistance to chemotherapy and radiation, and that downregulation may lead to sensitization to radiation and/or chemotherapy. We found that radiation and chemotherapy up-regulate Cav-1 expression, and that downregulating Cav-1 in multiple pancreatic cancer cell lines by small interfering RNA induces sensitization to chemotherapeutics such as gemcitabine and 5-fluorouracil. Furthermore, genetic knockdown causes reduction in tumor cell proliferation and colony formation. Knockdown of caveolin-1 also led to radiosensitization of tumor cells and resulted in delayed DNA repair as demonstrated by delayed pH2.AX foci resolution, increased mitotic catastrophe, as well as decreased formation of BRCA1 and DNAPK-cs foci in the nuclei of these cells. Furthermore, Cav-1 loss promoted cleavage of Caspase-9 and PARP after treatment with gemcitabine. These results indicate that knocking down Cav-1 in pancreatic cancer cells sensitizes them to radiation with delayed DNA damage repair and also to chemotherapeutics with the onset of apoptosis. In vivo, stable knockdown of Cav-1 attenuated the rate of tumor growth significantly compared to control tumors in subcutaneous xenograft pancreatic tumors. In summary we conclude that Cav-1 is responsible in promoting resistance to radiation and chemotherapy in pancreatic cancer cells and that downregulation of this protein leads to improved therapeutic response. Thus, it is possible that caveolin-1 may serve as a prognostic predictive biomarker in pancreatic cancer patients for response to radiation and chemotherapy. Citation Format: Moumita Chatterjee, Terence M. Williams. Caveolin-1 confers a multi-modality therapy resistance phenotype in pancreatic cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2014-4702