Lung Adenocarcinoma Tumor Origin: A Guide for Personalized Medicine.

Lung adenocarcinoma, the major form of lung cancer, is the deadliest cancer worldwide, due to its late diagnosis and its high heterogeneity. Indeed, lung adenocarcinoma exhibits pronounced inter- and intra-tumor heterogeneity cofounding precision medicine. Tumor heterogeneity is a clinical challenge driving tumor progression and drug resistance. Several key pieces of evidence demonstrated that lung adenocarcinoma results from the transformation of progenitor cells that accumulate genetic abnormalities. Thus, a better understanding of the cell of origin of lung adenocarcinoma represents an opportunity to unveil new therapeutic alternatives and stratify patient tumors. While the lung is remarkably quiescent during homeostasis, it presents an extensive ability to respond to injury and regenerate lost or damaged cells. As the lung is constantly exposed to potential insult, its regenerative potential is assured by several stem and progenitor cells. These can be induced to proliferate in response to injury as well as differentiate into multiple cell types. A better understanding of how genetic alterations and perturbed microenvironments impact progenitor-mediated tumorigenesis and treatment response is of the utmost importance to develop new therapeutic opportunities.

KRAS Addiction Promotes Cancer Cell Adaptation in Harsh Microenvironment Through Macropinocytosis.

KRAS is the most frequently mutated oncogene in cancer and despite intensive studies, attempts to develop effective therapies targeting KRAS or its downstream signaling have failed mostly due to the complexity of KRAS activation and function in cancer initiation and progression. Over the years, KRAS has been involved in several biological processes including cell survival, proliferation, and metabolism by promoting not only a favorable tumor environment but also a cell-microenvironment dialog to allow cancer cells to adapt to tumor microenvironment scarcity. One of the mechanisms involved in this adaption is KRAS-mediated macropinocytosis. Macropinocytosis is an evolutionarily conserved, large-scale, and nonselective form of endocytosis involving actin-driven cell membrane remodeling to engulf large amounts of extracellular fluids and proteins from the local environment. While macropinocytosis process has been known for decades, recent gain interest due to its regulation of KRAS-driven tumor growth in adverse microenvironments. By promoting extracellular protein and other macromolecules internalization, macropinocytosis provides a survival mechanism under nutrient scarce conditions and the potential for unrestricted tumor growth. Thus, a better understanding of macropinocytotic process is needed to develop alternative therapeutic strategies.

Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells.

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and ß1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles' heel for a significant and unique subset of GBM patients.

A small-molecule P2RX7 activator promotes anti-tumor immune responses and sensitizes lung tumor to immunotherapy.

Only a subpopulation of non-small cell lung cancer (NSCLC) patients responds to immunotherapies, highlighting the urgent need to develop therapeutic strategies to improve patient outcome. We develop a chemical positive modulator (HEI3090) of the purinergic P2RX7 receptor that potentiates αPD-1 treatment to effectively control the growth of lung tumors in transplantable and oncogene-induced mouse models and triggers long lasting antitumor immune responses. Mechanistically, the molecule stimulates dendritic P2RX7-expressing cells to generate IL-18 which leads to the production of IFN-γ by Natural Killer and CD4+ T cells within tumors. Combined with immune checkpoint inhibitor, the molecule induces a complete tumor regression in 80% of LLC tumor-bearing mice. Cured mice are also protected against tumor re-challenge due to a CD8-dependent protective response. Hence, combination treatment of small-molecule P2RX7 activator followed by immune checkpoint inhibitor represents a strategy that may be active against NSCLC.

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.

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.

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.

Integrin αvß3 drives slug activation and stemness in the pregnant and neoplastic mammary gland.

Although integrin αvß3 is linked to cancer progression, its role in epithelial development is unclear. Here, we show that αvß3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. Whereas αvß3 is a luminal progenitor marker in the virgin gland, we noted increased αvß3 expression in MaSCs at midpregnancy. Accordingly, mice lacking αvß3 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, αvß3-deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. Transforming growth factor ß2 (TGF-ß2) induced αvß3 expression, enhancing Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvß3 was necessary and sufficient for Slug activation, tumorsphere formation, and tumor initiation. Thus, pregnancy-associated MaSCs require a TGF-ß2/αvß3/Slug pathway, which may contribute to breast cancer progression and stemness.

Variety in the tumor microenvironment: integrin splicing regulates stemness.

Recently in Cell Reports, Goel et al. (2014) identified mechanisms underlying cellular heterogeneity in triple negative breast cancer. They find that expression of α6 integrin and its splice variants differs between epithelial and mesenchymal tumor cell subpopulations, the latter of which relies on VEGF signaling to promote cancer stem cell function.

An integrin ß3-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 α(v)ß3 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, α(v)ß3, 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, α(v)ß3 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 α(v)ß3 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.

APC(cdh1) mediates degradation of the oncogenic Rho-GEF Ect2 after mitosis.

BACKGROUND: Besides regulation of actin cytoskeleton-dependent functions, Rho GTPase pathways are essential to cell cycle progression and cell division. Rho, Rac and Cdc42 regulate G1 to S phase progression and are involved in cytokinesis. RhoA GDP/GTP cycling is required for normal cytokinesis and recent reports have shown that the exchange factor Ect2 and the GTPase activating protein MgcRacGAP regulate RhoA activity during mitosis. We previously showed that the transcription factors E2F1 and CUX1 regulate expression of MgcRacGAP and Ect2 as cells enter S-phase. METHODOLOGY/PRINCIPAL FINDINGS: We now report that Ect2 is subject to proteasomal degradation after mitosis, following ubiquitination by the APC/C complex and its co-activator Cdh1. A proper nuclear localization of Ect2 is necessary for its degradation. APC-Cdh1 assembles K11-linked poly-ubiquitin chains on Ect2, depending upon a stretch of ∼25 amino acid residues that contain a bi-partite NLS, a conventional D-box and two TEK-like boxes. Site-directed mutagenesis of target sequences generated stabilized Ect2 proteins. Furthermore, such degradation-resistant mutants of Ect2 were found to activate RhoA and subsequent signalling pathways and are able to transform NIH3T3 cells. CONCLUSIONS/SIGNIFICANCE: Our results identify Ect2 as a bona fide cell cycle-regulated protein and suggest that its ubiquitination-dependent degradation may play an important role in RhoA regulation at the time of mitosis. Our findings raise the possibility that the overexpression of Ect2 that has been reported in some human tumors might result not only from deregulated transcription, but also from impaired degradation.

CUX1 and E2F1 regulate coordinated expression of the mitotic complex genes Ect2, MgcRacGAP, and MKLP1 in S phase.

Rho GTPases are critical for mitosis progression and completion of cytokinesis. During mitosis, the GDP/GTP cycle of Rho GTPases is regulated by the exchange factor Ect2 and the GTPase activating protein MgcRacGAP which associates with the kinesin MKLP1 in the centralspindlin complex. We report here that expression of Ect2, MgcRacGAP, and MKLP1 is tightly regulated during cell cycle progression. These three genes share similar cell cycle-related signatures within their promoter regions: (i) cell cycle gene homology region (CHR) sites located at -20 to +40 nucleotides of their transcription start sites that are required for repression in G(1), (ii) E2F binding elements, and (iii) tandem repeats of target sequences for the CUX1 transcription factor. CUX1 and E2F1 bind these three promoters upon S-phase entry, as demonstrated by chromatin immunoprecipitation, and regulate transcription of these genes, as established using promoter-luciferase reporter constructs and expression of activated or dominant negative transcription factors. Overexpression of either E2F1 or CUX1 increased the levels of the endogenous proteins whereas small interfering RNA knockdown of E2F1 or use of a dominant negative E2F1 reduced their expression levels. Thus, CUX1, E2F, and CHR elements provide the transcriptional controls that coordinate induction of Ect2, MgcRacGAP, and MKLP1 in S phase, leading to peak expression of these interacting proteins in G(2)/M, at the time they are required to regulate cytokinesis.

Phosphoregulation of MgcRacGAP in mitosis involves Aurora B and Cdk1 protein kinases and the PP2A phosphatase.

MgcRacGAP, a Rho GAP essential to cytokinesis, works both as a Rho GTPase regulator and as a scaffolding protein. MgcRacGAP interacts with MKLP1 to form the centralspindlin complex and associates with the RhoGEF Ect2. The GAP activity of MgcRacGAP is regulated by Aurora B phosphorylation. We have isolated B56epsilon, a PP2A regulatory subunit, as a new MgcRacGAP partner. We report here that (i) MgcRacGAP is phosphorylated by Aurora B and Cdk1, (ii) PP2A dephosphorylates Aurora B and Cdk1 phosphorylated sites and (iii) inhibition of PP2A abrogates MgcRacGAP/Ect2 interaction. Therefore, PP2A may regulate cytokinesis by dephosphorylating MgcRacGAP and its interacting partners.