Jacqueline Lesperance

An integrin β 3 –KRAS–RalB complex drives tumour stemness and resistance to EGFR inhibition

Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, αvβ3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, αvβ3 expression and the resulting KRAS–RalB–NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

RBBP9: A tumor-associated serine hydrolase activity required for pancreatic neoplasia

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-β-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-β signaling. Finally, RBBP9-mediated suppression of TGF-β signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell–cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.

Oncogenic Ras/Src cooperativity in pancreatic neoplasia

Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5–8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDAs indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.

Galectin-3, a Druggable Vulnerability for KRAS-Addicted Cancers

Identifying the molecular basis for cancer cell dependence on oncogenes such as KRAS can provide new opportunities to target these addictions. Here, we identify a novel role for the carbohydrate-binding protein galectin-3 as a lynchpin for KRAS dependence. By directly binding to the cell surface receptor integrin αvβ3, galectin-3 gives rise to KRAS addiction by enabling multiple functions of KRAS in anchorage-independent cells, including formation of macropinosomes that facilitate nutrient uptake and ability to maintain redox balance. Disrupting αvβ3/galectin-3 binding with a clinically active drug prevents their association with mutant KRAS, thereby suppressing macropinocytosis while increasing reactive oxygen species to eradicate αvβ3-expressing KRAS-mutant lung and pancreatic cancer patient-derived xenografts and spontaneous tumors in mice. Our work reveals galectin-3 as a druggable target for KRAS-addicted lung and pancreas cancers, and indicates integrin αvβ3 as a biomarker to identify susceptible tumors.Significance: There is a significant unmet need for therapies targeting KRAS-mutant cancers. Here, we identify integrin αvβ3 as a biomarker to identify mutant KRAS-addicted tumors that are highly sensitive to inhibition of galectin-3, a glycoprotein that binds to integrin αvβ3 to promote KRAS-mediated activation of AKT. Cancer Discov; 7(12); 1464-79. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.

Proapoptotic PUMA targets stem-like breast cancer cells to suppress metastasis

Breast cancer cells with stem cell properties are key contributors to metastatic disease, and there remains a need to better understand and target these cells in human cancers. Here, we identified rare stem-like cells in patients’ tumors characterized by low levels of the proapoptotic molecule p53-upregulated modulator of apoptosis (PUMA) and showed that these cells play a critical role in tumor progression that is independent of clinical subtype. A signaling axis consisting of the integrin &agr;v&bgr;3, Src kinase, and the transcription factor Slug suppresses PUMA in these cells, promoting tumor stemness. We showed that genetic or pharmacological disruption of &agr;v&bgr;3/Src signaling drives PUMA expression, specifically depleting these stem-like tumor cells; increases their sensitivity to apoptosis; and reduces pulmonary metastasis, with no effect on primary tumor growth. Taken together, these findings point to PUMA as a key vulnerability of stem-like cells and suggest that pharmacological upregulation of PUMA via Src inhibition may represent a strategy to selectively target these cells in a wide spectrum of aggressive breast cancers.

Abstract 3321: PUMA expression targets αvβ3/Slug+ “stem-like” tumor cells to prevent breast cancer progression independent of subtype

Breast cancer cells bearing similarities to normal mammary stem cells represent some of the most aggressive tumors cells. Here, we show that αvβ3/Slug+ “stem-like” tumor cells are highly vulnerable to cell death induced by PUMA (p53-upregulated modulator of apoptosis). In human breast cancers, we were surprised to find that αvβ3/Slug+ cells represent a unique subset of tumor cells associated with progression that are present in all major histological subtypes. Analysis of “stem-like” cell lines showed that αvβ3 directs an EMT-independent role for Slug in suppressing PUMA expression and promoting tumor cell survival and stemness. In fact, αvβ3 is both necessary and sufficient for PUMA suppression and PUMA knockdown specifically rescued defective colony formation and tumor initiation caused by decreased β3 expression. Inducing PUMA expression with specific therapies selectively ablated αvβ3/Slug+ cells, preventing the formation of new colonies or tumors, with no effect on established disease. These new findings define an unexpected role for αvβ3/Slug-mediated PUMA suppression in driving stemness and suggest that inducing PUMA with specific therapies can selectively target “stem-like” breast cancer cells to prevent aggressive disease. Citation Format: Qi Sun, Jacqueline Lesperance, Jay S. Desgrosellier. PUMA expression targets αvβ3/Slug+ “stem-like” tumor cells to prevent breast cancer progression independent of subtype. [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 3321.

Abstract 3034: Integrin αvβ3 drives Slug activation and stemness in the pregnant and neoplastic mammary gland

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA While integrin αvβ3 expression is linked to breast cancer progression its role in epithelial development is unclear. Here, we show that αvβ3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. While αvβ3 is a luminal progenitor marker in the virgin gland, we noted increased αvβ3 expression in MaSCs at mid-pregnancy. Accordingly, mice lacking αvβ3 or expressing a signaling deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity and expression of Slug, a master regulator of MaSCs. Surprisingly, αvβ3 deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. TGFβ2, but not RANKL, induced αvβ3 leading to Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvβ3 was necessary and sufficient for Slug activation and tumorsphere formation. Thus, pregnancy-associated MaSCs require a TGFβ2/αvβ3/Slug pathway, which may also contribute to breast cancer progression. Citation Format: Jay S. Desgrosellier, Jacqueline Lesperance, Laetitia Seguin, Sanford J. Shattil, David A. Cheresh. Integrin αvβ3 drives Slug activation and stemness in the pregnant and neoplastic mammary gland. [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 3034. doi:10.1158/1538-7445.AM2014-3034

Abstract 1916: β3 integrin/KRAS/RalB complex drives tumor stemness and resistance to EGFR inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Integrin αvβ3 expression is a marker of tumor progression for a wide range of histologically distinct cancers, yet the molecular mechanism by which αvβ3 influences the growth and malignancy of cancer is poorly understood. Here, we reveal that integrin αvβ3, in the unligated state on lung, breast and pancreatic cancer cells, recruits KRAS and RalB to the plasma membrane, leading to the activation of TBK-1/c-Rel. These events are both necessary and sufficient to promote tumor initiation, anchorage-independence, self-renewal, as well as resistance to nutrient deprivation and treatment with receptor tyrosine kinase (RTK) inhibitors such as erlotinib. Genetic or pharmacological inhibition of RalB or its effectors suppress tumor initiation, self-renewal, and re-sensitizes tumors to RTK inhibition. These findings not only identify ανβ3 as a marker/driver of tumor stemness but also provide a strategy to overcome tumor resistance to RTK Inhibition. Citation Format: Laetitia Jocelyne Seguin, Shumei Kato, Aleksandra Franovic, Maria Fernanda Camargo, Katrhyn Elliott, Mayra Yebra, Jacqueline Lesperance, Ainhoa Mielgo, Jay Desgrosellier, Sudarshan Anand, Sara Weis, David Cheresh. β3 integrin/KRAS/RalB complex drives tumor stemness and resistance to EGFR inhibition. [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 1916. doi:10.1158/1538-7445.AM2014-1916

Abstract A036: Integrin αvβ3 drives the mammary stem cell state during pregnancy

Epithelial stem/progenitor cell properties are associated with aggressive carcinomas. The integrin αvβ3 promotes tumor progression and metastasis in a diverse array of human carcinomas, and may be integral to epithelial stem/progenitor cell behavior. Here we reveal a critical role for αvβ3 in adult mammary stem cells (MaSCs) during pregnancy. While αvβ3 is a luminal progenitor marker in the virgin mammary gland, we noted a dramatic increase in αvβ3 expression in the MaSC pool at mid-pregnancy associated with enhanced repopulating potential. Similar to pregnancy, we noted that TGFβ, and not RANKL, was sufficient to drive αvβ3 expression enhancing MaSC activity. Accordingly, mice lacking β3 or expressing a signaling deficient form of this receptor showed defective mammary gland morphogenesis during pregnancy associated with decreased MaSC clonogenicity, expansion and outgrowth potential. Surprisingly, these mice displayed normal development of the virgin gland with no effect on luminal progenitors. These findings support a role for αvβ3 as a critical determinant of the MaSC state during pregnancy and suggest a related role for this receptor in regulating stem-like behavior in human breast cancers. Citation Format: Jay Desgrosellier, Jacqueline Lesperance, Laetitia Seguin, Sanford Shattil, David Cheresh. Integrin αvβ3 drives the mammary stem cell state during pregnancy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A036.

Abstract C78: Integrin αvβ3 drives stemness in the pregnant and neoplastic mammary gland

Carcinoma cell expression of the integrin αvβ3 is associated with enhanced tumorigenicity and metastasis in a diverse array of human tumors. Here, we reveal αvβ3 expression in a subset of CD44+CD24-/low cancer stem cells in human breast tumor biopsies. In fact, αvβ3 is both necessary and sufficient to account for stem-like properties in breast tumor cells, suggesting that αvβ3 may contribute to mammary stem (MaSC)/progenitor cell behavior. While αvβ3 is a luminal progenitor marker in the virgin murine mammary gland, we noted a dramatic, but transient, increase in αvβ3 levels that defined the mammary stem cell pool at mid-pregnancy. Genetic deletion of β3 surprisingly had no effect on luminal progenitor behavior or ductal morphogenesis. In contrast, β3 was specifically required for MaSC clonogenicity and alveologenesis at mid-pregnancy, yet had no influence on MaSCs in the virgin gland. These findings reveal an unexpected role for αvβ3 in driving mammary stemness during pregnancy that may be related to the stem-like properties, and high metastatic potential associated with αvβ3 expression in human breast cancers. Citation Format: Jay Desgrosellier, Jacqueline Lesperance, Laetitia Seguin, David Cheresh. Integrin αvβ3 drives stemness in the pregnant and neoplastic mammary gland. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C78.