Laetitia Seguin

Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance

Interactions between cancer cells and their surroundings can trigger essential signaling cues that determine cell fate and influence the evolution of the malignant phenotype. As the primary receptors involved in cell-matrix adhesion, integrins present on the surface of tumor and stromal cells have a profound impact on the ability to survive in specific locations, but in some cases, these receptors can also function in the absence of ligand binding to promote stemness and survival in the presence of environmental and therapeutic stresses. Understanding how integrin expression and function is regulated in this context will enable the development of new therapeutic approaches to sensitize tumors to therapy and suppress their metastatic phenotype.

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.

A MEK-independent role for CRAF in mitosis and tumor progression.

RAF kinases regulate cell proliferation and survival and can be dysregulated in tumors. The role of RAF in cell proliferation has been linked to its ability to activate mitogen-activated protein kinase kinase 1 (MEK) and mitogen-activated protein kinase 1 (ERK). Here we identify a MEK-independent role for RAF in tumor growth. Specifically, in mitotic cells, CRAF becomes phosphorylated on Ser338 and localizes to the mitotic spindle of proliferating tumor cells in vitro as well as in murine tumor models and in biopsies from individuals with cancer. Treatment of tumors with allosteric inhibitors, but not ATP-competitive RAF inhibitors, prevents CRAF phosphorylation on Ser338 and localization to the mitotic spindle and causes cell-cycle arrest at prometaphase. Furthermore, we identify phospho-Ser338 CRAF as a potential biomarker for tumor progression and a surrogate marker for allosteric RAF blockade. Mechanistically, CRAF, but not BRAF, associates with Aurora kinase A (Aurora-A) and Polo-like kinase 1 (Plk1) at the centrosomes and spindle poles during G2/M. Indeed, allosteric or genetic inhibition of phospho-Ser338 CRAF impairs Plk1 activation and accumulation at the kinetochores, causing prometaphase arrest, whereas a phospho-mimetic Ser338D CRAF mutant potentiates Plk1 activation, mitosis and tumor progression in mice. These findings show a previously undefined role for RAF in tumor progression beyond the RAF-MEK-ERK paradigm, opening new avenues for targeting RAF in cancer.

Kinase-independent role for CRAF-driving tumour radioresistance via CHK2

Although oncology therapy regimens commonly include radiation and genotoxic drugs, tumour cells typically develop resistance to these interventions. Here we report that treatment of tumours with ionizing radiation or genotoxic drugs drives p21-activated kinase 1 (PAK1)-mediated phosphorylation of CRAF on Serine 338 (pS338) triggering a kinase-independent mechanism of DNA repair and therapeutic resistance. CRAF pS338 recruits CHK2, a cell cycle checkpoint kinase involved in DNA repair, and promotes CHK2 phosphorylation/activation to enhance the tumour cell DNA damage response. Accordingly, a phospho-mimetic mutant of CRAF (S338D) is sufficient to induce the CRAF/CHK2 association enhancing tumour radioresistance, while an allosteric CRAF inhibitor sensitizes tumour cells to ionizing radiation or genotoxic drugs. Our findings establish a role for CRAF in the DNA damage response that is independent from its canonical function as a kinase.

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.

Glioblastomas require integrin αvβ3/PAK4 signaling to escape senescence.

Integrin αvβ3 has been implicated as a driver of aggressive and metastatic disease, and is upregulated during glioblastoma progression. Here, we demonstrate that integrin αvβ3 allows glioblastoma cells to counteract senescence through a novel tissue-specific effector mechanism involving recruitment and activation of the cytoskeletal regulatory kinase PAK4. Mechanistically, targeting either αvβ3 or PAK4 led to emergence of a p21-dependent, p53-independent cell senescence phenotype. Notably, glioblastoma cells did not exhibit a similar requirement for either other integrins or additional PAK family members. Moreover, αvβ3/PAK4 dependence was not found to be critical in epithelial cancers. Taken together, our findings established that glioblastomas are selectively addicted to this pathway as a strategy to evade oncogene-induced senescence, with implications that inhibiting the αvβ3-PAK4 signaling axis may offer novel therapeutic opportunities to target this aggressive cancer.

Abstract 3966: Targeting integrin αvβ3-expressing cancer stem cells to manipulate tumor-associated macrophages

Tumor associated macrophages are involved in regulation of cancer growth and aggressiveness. Whereas M1 macrophages trigger an inflammatory response and inhibit tumor growth, M2 macrophages secrete pro-tumor cytokines into the microenvironment to support tumor progression. A macrophage switch from M1 to M2 has been associated with lung cancer progression, and cancer stem cells have been implicated as a driver of this reprogramming. We recently reported that integrin αvβ3 expression is induced on lung adenocarcinoma cells during drug resistance and is both necessary and sufficient to reprogram these tumors to a stem-like state. Given the role that cancer stem cells play in switching M1 to M2 macrophages, we asked whether αvβ3 expression on lung adenocarcinoma cells account for this macrophage conversion. The M1/M2 macrophage ratio in αvβ3-positive tumors was markedly decreased relative to tumors lacking αvβ3. We next treated mice bearing αvβ3-positive tumors with a monoclonal antibody (LM609) targeting this receptor to assess its ability to alter the macrophage phenotype within these tumors. LM609 was able to selectively eliminate the αvβ3-positive cancer stem cells via antibody-dependent cell-mediated cytotoxicity (ADCC), and this not only increased the M1 macrophage population, but also markedly enhanced the sensitivity of these tumors to the effects of therapy. These findings reveal that αvβ3-expressing cancer stem cells favor the pro-tumor M2 macrophage phenotype. Eliminating αvβ3-positive cancer stem cells via ADCC serves to both increase pro-inflammatory macrophages within the tumor microenvironment and prolong tumor sensitivity to therapy. Citation Format: Hiromi I. Wettersten, Toshiyuki Minami, Megan M. Kaneda, Laetitia Seguin, Judith A. Varner, Sara M. Weis, David A. Cheresh. Targeting integrin αvβ3-expressing cancer stem cells to manipulate tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3966. doi:10.1158/1538-7445.AM2017-3966

Abstract 3067: Galectin-3, a target for KRAS-addicted lung cancer

KRAS-mutant cancers are notoriously therapy-resistant. While responsible for oncogenesis, mutant KRAS or the sustained activation of KRAS-driven effectors may or may not be required throughout the course of cancer progression. Thus, understanding which tumors remain dependent on oncogenic KRAS could lead to the identification of unique vulnerabilities and opportunities to treat them. We previously discovered that the carbohydrate-binding Galectin-3 (Gal-3) brings together KRAS with integrin αvβ3 at the cell membrane in epithelial cancer cells to promote tumor progression. Here, we show that by directly binding to the cell surface receptor integrin αvβ3, Gal-3 drives addiction to oncogenic KRAS by enhancing macropinocytosis and reducing mitochondrial reactive oxygen species (ROS). This pathway drives increased expression of SOD2, a superoxide dismutase that clears mitochondrial ROS to protect against cell death. Targeting Gal-3 with a clinically active drug decreases macropinocytosis and increases ROS to eradicate KRAS-addicted lung cancer in patient-derived xenografts and spontaneous KRAS-driven lung cancer in mice. Our work reveals Gal-3 as a druggable target for KRAS-addicted lung cancer, and indicates integrin αvβ3 as a biomarker to identify this dependence. Citation Format: Laetitia Seguin, Maria F. Camargo, Hiromi I. Wettersten, Shumei Kato, Tami von Schalscha, Kathryn C. Elliott, Sara M. Weis, David A. Cheresh. Galectin-3, a target for KRAS-addicted lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2017-3067

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 4240: Targeting Galectin-3 to reverse tumor stemness and drug resistance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have recently linked integrin αvβ3 expression to metastatic potential, stemness, and therapy resistance due to its capacity to couple to KRAS. This leads to the induction of RalB/TBK-1 and the activation of NFκB to shift tumor cells to a stem-like phenotype. To understand how integrin αvβ3 couples to KRAS, we generated β3 domain swap mutants containing either the intracellular or extracellular domains of β1 integrin. Each chimera partially reduced KRAS association with αvβ3, suggesting interactions on each side of the plasma membrane may contribute. Since the short β3 cytoplasmic tail has no putative KRAS binding regions, we considered Galectin-3 as a putative scaffolding partner to facilitate the αvβ3/KRAS association due to its reported binding to both KRAS and integrins. Indeed, treating cells with GCS-100, a complex polysaccharide that has the ability to bind to and block the effects of Galectin-3, decreased Galectin-3 surface expression, αvβ3 clustering, and αvβ3/KRAS association. Exposing cells to this drug also decreased tumor stem properties. Our observations provide a molecular basis for the αvβ3/KRAS/Galectin-3 complex and highlight the potential for Galectin-3 blockade as a therapeutic approach to reverse the aggressive behavior of αvβ3-expressing tumors. Citation Format: Shumei Kato, Laetitia Seguin, Aleksandra Franovic, Hisashi Kato, Maria Camargo, Jay Desgrosellier, Sudarshan Anand, Sara Weis, Sanford Shattil, David A. Cheresh. Targeting Galectin-3 to reverse tumor stemness and drug resistance. [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 4240. doi:10.1158/1538-7445.AM2014-4240