Integrin αvß3 Upregulation in Response to Nutrient Stress Promotes Lung Cancer Cell Metabolic Plasticity.

Cancer stem/tumor-initiating cells display stress tolerance and metabolic flexibility to survive in a harsh environment with limited nutrient and oxygen availability. The molecular mechanisms underlying this phenomenon could provide targets to prevent metabolic adaptation and halt cancer progression. Here, we showed in cultured cells and live human surgical biopsies of non-small cell lung cancer that nutrient stress drives the expression of the epithelial cancer stem cell marker integrin αvß3 via upregulation of the ß3 subunit, resulting in a metabolic reprogramming cascade that allows tumor cells to thrive despite a nutrient-limiting environment. Although nutrient deprivation is known to promote acute, yet transient, activation of the stress sensor AMP-activated protein kinase (AMPK), stress-induced αvß3 expression via Src activation unexpectedly led to secondary and sustained AMPK activation. This resulted in the nuclear localization of peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α) and upregulation of glutamine metabolism, the tricarboxylic acid cycle, and oxidative phosphorylation. Pharmacological or genetic targeting of this axis prevented lung cancer cells from evading the effects of nutrient stress, thereby blocking tumor initiation in mice following orthotopic implantation of lung cancer cells. These findings reveal a molecular pathway driven by nutrient stress that results in cancer stem cell reprogramming to promote metabolic flexibility and tumor initiation. SIGNIFICANCE: Upregulation of integrin αvß3, a cancer stem cell marker, in response to nutrient stress activates sustained AMPK/PGC1α signaling that induces metabolic reprogramming in lung cancer cells to support their survival. See related commentary by Rainero, p. 1543.

Targeting the αVß3/NgR2 pathway in neuroendocrine prostate cancer.

Highly aggressive, metastatic, neuroendocrine prostate cancer, which typically develops from prostate cancer cells acquiring resistance to androgen deprivation therapy, is associated with limited treatment options and hence poor prognosis. We have previously demonstrated that the αVß3 integrin is over-expressed in neuroendocrine prostate cancer. We now show that LM609, a monoclonal antibody that specifically targets the human αVß3 integrin, hinders the growth of neuroendocrine prostate cancer patient-derived xenografts in vivo. Our group has recently identified a novel αVß3 integrin binding partner, NgR2, responsible for regulating the expression of neuroendocrine markers and for inducing neuroendocrine differentiation in prostate cancer cells. Through in vitro functional assays, we here demonstrate that NgR2 is crucial in promoting cell adhesion to αVß3 ligands. Moreover, we describe for the first time co-fractionation of αVß3 integrin and NgR2 in small extracellular vesicles derived from metastatic prostate cancer patients' plasma. These prostate cancer patient-derived small extracellular vesicles have a functional impact on human monocytes, increasing their adhesion to fibronectin. The monocytes incubated with small extracellular vesicles do not show an associated change in conventional polarization marker expression and appear to be in an early stage that may be defined as "adhesion competent". Overall, these findings allow us to better understand integrin-directed signaling and cell-cell communication during cancer progression. Furthermore, our results pave the way for new diagnostic and therapeutic perspectives for patients affected by neuroendocrine prostate cancer.

Upregulation of fibronectin and its integrin receptors - an adaptation to isolation stress that facilitates tumor initiation.

Tumor initiation at either primary or metastatic sites is an inefficient process in which tumor cells must fulfill a series of conditions. One critical condition involves the ability of individual tumor-initiating cells to overcome 'isolation stress', enabling them to survive within harsh isolating microenvironments that can feature nutrient stress, hypoxia, oxidative stress and the absence of a proper extracellular matrix (ECM). In response to isolation stress, tumor cells can exploit various adaptive strategies to develop stress tolerance and gain stemness features. In this Opinion, we discuss how strategies such as the induction of certain cell surface receptors and deposition of ECM proteins enable tumor cells to endure isolation stress, thereby gaining tumor-initiating potential. As examples, we highlight recent findings from our group demonstrating how exposure of tumor cells to isolation stress upregulates the G-protein-coupled receptor lysophosphatidic acid receptor 4 (LPAR4), its downstream target fibronectin and two fibronectin-binding integrins, α5ß1 and αvß3. These responses create a fibronectin-rich niche for tumor cells, ultimately driving stress tolerance, cancer stemness and tumor initiation. We suggest that approaches to prevent cancer cells from adapting to stress by suppressing LPAR4 induction, blocking its downstream signaling or disrupting fibronectin-integrin interactions hold promise as potential strategies for cancer treatment.

Tumor-initiating cells establish a niche to overcome isolation stress.

While the tumor microenvironment is a critical contributor to cancer progression, early steps of tumor initiation and metastasis also rely on the ability of individual tumor cells to survive and thrive at locations where tumor stroma or immune infiltration has yet to be established. In this opinion article, we use the term 'isolation stress' to broadly describe the challenges that individual tumor cells must overcome during the initiation and expansion of the primary tumor beyond permissive boundaries and metastatic spread into distant sites, including a lack of cell-cell contact, adhesion to protumor extracellular matrix proteins, and access to nutrients, oxygen, and soluble factors that support growth. In particular, we highlight the ability of solitary tumor cells to autonomously generate a specialized fibronectin-enriched extracellular matrix to create their own pericellular niche that supports tumor initiation. Cancer cells that can creatively evade the effects of isolation stress not only become more broadly stress tolerant, they also tend to show enhanced stemness, drug resistance, tumor initiation, and metastasis.

Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to promote an ECM-enriched niche and support tumour initiation.

Defining drivers of tumour initiation can provide opportunities to control cancer progression. Here we report that lysophosphatidic acid receptor 4 (LPAR4) becomes transiently upregulated on pancreatic cancer cells exposed to environmental stress or chemotherapy where it promotes stress tolerance, drug resistance, self-renewal and tumour initiation. Pancreatic cancer cells gain LPAR4 expression in response to stress by downregulating a tumour suppressor, miR-139-5p. Even in the absence of exogenous lysophosphatidic acid, LPAR4-expressing tumour cells display an enrichment of extracellular matrix genes that are established drivers of cancer stemness. Mechanistically, upregulation of fibronectin via an LPAR4/AKT/CREB axis is indispensable for LPAR4-induced tumour initiation and stress tolerance. Moreover, ligation of this fibronectin-containing matrix via integrins α5ß1 or αVß3 can transfer stress tolerance to LPAR4-negative cells. Therefore, stress- or drug-induced LPAR4 enhances cell-autonomous production of a fibronectin-rich extracellular matrix, allowing cells to survive 'isolation stress' and compensate for the absence of stromal-derived factors by creating their own tumour-initiating niche.

Zika Virus Targets Glioblastoma Stem Cells through a SOX2-Integrin αvß5 Axis.

Zika virus (ZIKV) causes microcephaly by killing neural precursor cells (NPCs) and other brain cells. ZIKV also displays therapeutic oncolytic activity against glioblastoma (GBM) stem cells (GSCs). Here we demonstrate that ZIKV preferentially infected and killed GSCs and stem-like cells in medulloblastoma and ependymoma in a SOX2-dependent manner. Targeting SOX2 severely attenuated ZIKV infection, in contrast to AXL. As mechanisms of SOX2-mediated ZIKV infection, we identified inverse expression of antiviral interferon response genes (ISGs) and positive correlation with integrin αv (ITGAV). ZIKV infection was disrupted by genetic targeting of ITGAV or its binding partner ITGB5 and by an antibody specific for integrin αvß5. ZIKV selectively eliminated GSCs from species-matched human mature cerebral organoids and GBM surgical specimens, which was reversed by integrin αvß5 inhibition. Collectively, our studies identify integrin αvß5 as a functional cancer stem cell marker essential for GBM maintenance and ZIKV infection, providing potential brain tumor therapy.

Arming Tumor-Associated Macrophages to Reverse Epithelial Cancer Progression.

Tumor-associated macrophages (TAM) are highly expressed within the tumor microenvironment of a wide range of cancers, where they exert a protumor phenotype by promoting tumor cell growth and suppressing antitumor immune function. Here, we show that TAM accumulation in human and mouse tumors correlates with tumor cell expression of integrin αvß3, a known driver of epithelial cancer progression and drug resistance. A monoclonal antibody targeting αvß3 (LM609) exploited the coenrichment of αvß3 and TAMs to not only eradicate highly aggressive drug-resistant human lung and pancreas cancers in mice, but also to prevent the emergence of circulating tumor cells. Importantly, this antitumor activity in mice was eliminated following macrophage depletion. Although LM609 had no direct effect on tumor cell viability, it engaged macrophages but not natural killer (NK) cells to induce antibody-dependent cellular cytotoxicity (ADCC) of αvß3-expressing tumor cells despite their expression of the CD47 "don't eat me" signal. In contrast to strategies designed to eliminate TAMs, these findings suggest that anti-αvß3 represents a promising immunotherapeutic approach to redirect TAMs to serve as tumor killers for late-stage or drug-resistant cancers. SIGNIFICANCE: Therapeutic antibodies are commonly engineered to optimize engagement of NK cells as effectors. In contrast, LM609 targets αvß3 to suppress tumor progression and enhance drug sensitivity by exploiting TAMs to trigger ADCC.

Targeting the perivascular niche sensitizes disseminated tumour cells to chemotherapy.

The presence of disseminated tumour cells (DTCs) in bone marrow is predictive of poor metastasis-free survival of patients with breast cancer with localized disease. DTCs persist in distant tissues despite systemic administration of adjuvant chemotherapy. Many assume that this is because the majority of DTCs are quiescent. Here, we challenge this notion and provide evidence that the microenvironment of DTCs protects them from chemotherapy, independent of cell cycle status. We show that chemoresistant DTCs occupy the perivascular niche (PVN) of distant tissues, where they are protected from therapy by vascular endothelium. Inhibiting integrin-mediated interactions between DTCs and the PVN, driven partly by endothelial-derived von Willebrand factor and vascular cell adhesion molecule 1, sensitizes DTCs to chemotherapy. Importantly, chemosensitization is achieved without inducing DTC proliferation or exacerbating chemotherapy-associated toxicities, and ultimately results in prevention of bone metastasis. This suggests that prefacing adjuvant therapy with integrin inhibitors is a viable clinical strategy to eradicate DTCs and prevent metastasis.

Integrin CD11b activation drives anti-tumor innate immunity.

Myeloid cells are recruited to damaged tissues where they can resolve infections and tumor growth or stimulate wound healing and tumor progression. Recruitment of these cells is regulated by integrins, a family of adhesion receptors that includes integrin CD11b. Here we report that, unexpectedly, integrin CD11b does not regulate myeloid cell recruitment to tumors but instead controls myeloid cell polarization and tumor growth. CD11b activation promotes pro-inflammatory macrophage polarization by stimulating expression of microRNA Let7a. In contrast, inhibition of CD11b prevents Let7a expression and induces cMyc expression, leading to immune suppressive macrophage polarization, vascular maturation, and accelerated tumor growth. Pharmacological activation of CD11b with a small molecule agonist, Leukadherin 1 (LA1), promotes pro-inflammatory macrophage polarization and suppresses tumor growth in animal models of murine and human cancer. These studies identify CD11b as negative regulator of immune suppression and a target for cancer immune therapy.

Pseudopodium-enriched atypical kinase 1 mediates angiogenesis by modulating GATA2-dependent VEGFR2 transcription.

PEAK1 is a newly described tyrosine kinase and scaffold protein that transmits integrin-mediated extracellular matrix (ECM) signals to facilitate cell movement and growth. While aberrant expression of PEAK1 has been linked to cancer progression, its normal physiological role in vertebrate biology is not known. Here we provide evidence that PEAK1 plays a central role in orchestrating new vessel formation in vertebrates. Deletion of the PEAK1 gene in zebrafish, mice, and human endothelial cells (ECs) induced severe defects in new blood vessel formation due to deficiencies in EC proliferation, survival, and migration. Gene transcriptional and proteomic analyses of PEAK1-deficient ECs revealed a significant loss of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA and protein expression, as well as downstream signaling to its effectors, ERK, Akt, and Src kinase. PEAK1 regulates VEGFR2 expression by binding to and increasing the protein stability of the transcription factor GATA-binding protein 2 (GATA2), which controls VEGFR2 transcription. Importantly, PEAK1-GATA2-dependent VEGFR2 expression is mediated by EC adhesion to the ECM and is required for breast cancer-induced new vessel formation in mice. Also, elevated expression of PEAK1 and VEGFR2 mRNA are highly correlated in many human cancers including breast cancer. Together, our findings reveal a novel PEAK1-GATA2-VEGFR2 signaling axis that integrates cell adhesion and growth factor cues from the extracellular environment necessary for new vessel formation during vertebrate development and cancer.

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 αvß3, Src kinase, and the transcription factor Slug suppresses PUMA in these cells, promoting tumor stemness. We showed that genetic or pharmacological disruption of αvß3/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.

Glut3 Addiction Is a Druggable Vulnerability for a Molecularly Defined Subpopulation of Glioblastoma.

While molecular subtypes of glioblastoma (GBM) are defined using gene expression and mutation profiles, we identify a unique subpopulation based on addiction to the high-affinity glucose transporter, Glut3. Although Glut3 is a known driver of a cancer stem cell phenotype, direct targeting is complicated by its expression in neurons. Using established GBM lines and patient-derived stem cells, we identify a subset of tumors within the "proneural" and "classical" subtypes that are addicted to aberrant signaling from integrin αvß3, which activates a PAK4-YAP/TAZ signaling axis to enhance Glut3 expression. This defined subpopulation of GBM is highly sensitive to agents that disrupt this pathway, including the integrin antagonist cilengitide, providing a targeted therapeutic strategy for this unique subset of GBM tumors.

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 αvß3, 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 αvß3/galectin-3 binding with a clinically active drug prevents their association with mutant KRAS, thereby suppressing macropinocytosis while increasing reactive oxygen species to eradicate αvß3-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 αvß3 as a biomarker to identify susceptible tumors.Significance: There is a significant unmet need for therapies targeting KRAS-mutant cancers. Here, we identify integrin αvß3 as a biomarker to identify mutant KRAS-addicted tumors that are highly sensitive to inhibition of galectin-3, a glycoprotein that binds to integrin αvß3 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.

Treatment of rats with the JAK-2 inhibitor fedratinib does not lead to experimental Wernicke's encephalopathy.

Recent clinical trials suggest that patients with myelofibrosis can develop Wernicke's encephalopathy (WE) when treated with fedratinib, a specific Janus kinase-2 (JAK-2) inhibitor. To investigate this issue, we have examined (1) if fedratinib can produce or alter the course of this disorder, (2) its effects on thiamine-dependent enzyme activity and thiamine status, and (3) its influence on the uptake of thiamine. Animals administered fedratinib for 28days at a comparable dose used to treat human cases of myelofibrosis showed no evidence of clinical signs of thiamine deficiency (TD). Rats treated with a combination of fedratinib and TD exhibited no neurological differences in their progress to the symptomatic stage when compared to thiamine-deficient animals only. Treatment with the JAK-2 inhibitor did not compromise erythrocyte transketolase activity, and thiamine status was not affected in a major way unlike animals with TD. In addition, treatment of cultured astrocytes with fedratinib did not diminish the uptake of thiamine into these cells. Our findings suggest that treatment with fedratinib does not lead to or alter the progress of TD, and do not support the notion that administration of this JAK-2 inhibitor directly results in the development of WE due to inhibition of thiamine transport. Known adverse effects of fedratinib involving compromised gastrointestinal function may be an important indirect contributing factor to previously reported cases of WE in patients with myelofibrosis.

MicroRNA regulation of endothelial TREX1 reprograms the tumour microenvironment.

Rather than targeting tumour cells directly, elements of the tumour microenvironment can be modulated to sensitize tumours to the effects of therapy. Here we report a unique mechanism by which ectopic microRNA-103 can manipulate tumour-associated endothelial cells to enhance tumour cell death. Using gain-and-loss of function approaches, we show that miR-103 exacerbates DNA damage and inhibits angiogenesis in vitro and in vivo. Local, systemic or vascular-targeted delivery of miR-103 in tumour-bearing mice decreased angiogenesis and tumour growth. Mechanistically, miR-103 regulation of its target gene TREX1 in endothelial cells governs the secretion of pro-inflammatory cytokines into the tumour microenvironment. Our data suggest that this inflammatory milieu may potentiate tumour cell death by supporting immune activation and inducing tumour expression of Fas and TRAIL receptors. Our findings reveal miR-mediated crosstalk between vasculature and tumour cells that can be exploited to improve the efficacy of chemotherapy and radiation.

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.

Glioblastomas require integrin αvß3/PAK4 signaling to escape senescence.

Integrin αvß3 has been implicated as a driver of aggressive and metastatic disease, and is upregulated during glioblastoma progression. Here, we demonstrate that integrin αvß3 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 αvß3 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, αvß3/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 αvß3-PAK4 signaling axis may offer novel therapeutic opportunities to target this aggressive cancer.

hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature.

To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC), not the neural tube (NT). Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC)-secreted nitric oxide (NO) and direct contact with vascular smooth muscle cells (VSMCs) via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.