Oncogene

The non-canonical translation of specific mRNAs has been implicated in oncogenesis and cancer progression. We previously identified eukaryotic Initiation Factor 5B (eIF5B) as a key factor in Internal Ribosome Entry Site (IRES)-mediated translation of a subset of mRNAs encoding anti-apoptotic proteins. Here, we demonstrate that EIF5B is predominantly expressed in cancer cells compared to other cell types in the Oral Squamous Cell Carcinoma (OSCC) microenvironment. Higher EIF5B mRNA and protein expression are associated with poor patient outcomes. We show that eIF5B depletion in OSCC cells blunted pro-growth, pro-inflammatory, and pro-angiogenic signaling pathways and significantly increased TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death. This is achieved through decreased translation of mRNAs encoding critical factors associated with OSCC pathophysiology. Importantly, the level of interaction of eIF5B with tRNAiMet was significantly higher in OSCC cells compared to non-cancerous fibroblasts. This suggests that OSCC cells (but not non-cancerous fibroblasts) rely heavily on eIF5B for translation initiation. In an in vivo flank xenograft model using nude mice, eIF5B knockdown in UMSCC-29 cells led to a significant reduction in tumor volume compared to control tumors. Also, the immunohistochemical analysis of the xenografted tumor sections demonstrated decreased staining intensity of critical factors associated with OSCC pathophysiology in eIF5B-depleted tumors relative to controls. Collectively, our data demonstrate that OSCC cells are uniquely dependent on eIF5B-tRNA interactions to sustain translation of pro-survival mRNAs. Targeting eIF5B disrupts these oncogenic programs, sensitizing OSCC cells to apoptosis and suppressing pro-angiogenic and pro-growth signaling.

Expression of pro-lymphangiogenic vascular endothelial growth factor C (VEGF-C) in primary tumors correlates with the occurrence of proximal lymph node metastasis in most solid cancer types. However, the role of VEGF-C in regulating tumor cell dissemination to distant organs is currently unclear. Perivascular tumor-associated macrophages (TAMs) are key regulators of hematogenous cancer cell spreading, forming tumor microenvironment of metastasis (TMEM) doorways for breast cancer cells to intravasate tumor blood vessels and fuel distant metastases. Using an experimental breast cancer (BC) model, we show here that TAMs expressing VEGF-C decrease cancer cell dissemination to the lung while enhancing lymph node metastasis. These TAMs express podoplanin and associate with normalized tumor blood vessels expressing VEGFR3. Further clinical data reveal that VEGF-C+ TAMs correlate inversely with malignant grade and with the occurrence of TMEM complexes in a cohort of BC patients. Thus, our study displays an apparently paradoxical role of VEGF-C expressing TAMs in redirecting cancer cells to preferentially disseminate to the lymph nodes, at least in part, by normalizing tumor blood vessels and promoting lymphangiogenesis.

Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer (1, 2). Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhoms, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17 (3, 4). Here we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing iRhom2 as a central component of a positive feedback loop in lung cancer cells. Overall, the cytoplasmic domain of iRhom2 is a critical component of KRAS-induced oncogenesis of lung cancer cells. Both ADAM17 and iRhom2 have also been implicated in a wide range of other cancers (5-10), so the mechanism we have revealed may also have wider oncogenic significance.

Integrin 6{beta}4 is highly expressed in triple negative breast cancer (TNBC) and drives aggressiveness by stimulating proliferation, angiogenesis, cell migration, invasion and metastasis. Signaling from this integrin stimulates DNA repair and apoptosis resistance, suggesting that it could contribute to therapeutic resistance. Upon testing this hypothesis, we found that integrin 6{beta}4 signaling promoted a three-fold greater sensitivity to cisplatin but exhibited no difference in response to other chemotherapies tested. Mechanistic investigations revealed that integrin 6{beta}4 stimulated quicker and higher amplitude of activation of ATM, Chk2, p53, and 53BP1, which required the integrin {beta}4 signaling domain. Genetic manipulation of gene expression demonstrated that mutant p53 cooperated with integrin 6{beta}4 for cisplatin sensitivity and was necessary for downstream phosphorylation of 53BP1 and enhanced ATM activation. Additionally, we discovered that integrin 6{beta}4 preferentially activated DNA-PKc in response to cisplatin, which led to formation of DNA-PKc-p53 complexes and 53BP1 activation. As a result, integrin 6{beta}4 shifted double strand break repair from homologous recombination (HR) to non-homologous end joining (NHEJ). In summary, we discovered a novel function of integrin 6{beta}4 in switching DSB repair from HR to NHEJ that results in cisplatin sensitivity in TNBC.

KRAS is commonly mutated in lung, colorectal, and pancreatic cancers. Small molecule inhibitors targeting KRAS with distinct mechanisms-of-action and variable specificities have entered the clinic, but a comprehensive view of their effect on the RAS signaling network has not been reported. Here, we describe the impact of RAS inhibition on the KRAS protein interactome using Proximity-dependent Biotinylation Identification. Two inhibitors were used: panRAS-ON, which forms a ternary complex between cyclophilin and RAS in the GTP bound state; and panKRAS-off, which binds specifically to the KRAS switch II pocket. RAS inhibitors were found to significantly alter 16.5% of proteins in proximity to KRAS. Despite their distinct mechanisms-of-action, the KRAS inhibitors induced highly correlated changes in the KRAS interactomes. Among proteins in close proximity to KRAS, Afadin was found to be highly regulated by RAS inhibition. AFDN has been characterized as a key regulator of cell motility, invasion, and metastasis. Analysis of AFDN phosphorylation revealed that AKT only partially modulates p-Ser1718, while inhibition of KRAS is sufficient to abolish EGF-mediated AFDN phosphorylation. Knockdown of AFDN in a KRAS-driven non-small cell lung cancer model abolished chemotaxis in a transwell migration assay and disrupted directional movement in an EGF-driven wound healing model. These results suggest that KRAS is a central node in regulating growth factor induced cell migration, and that KRAS inhibition plays a broader role than MAPK-mediated cell proliferation and survival.

Cancer-testis antigens (CTAs) are germ cell-restricted proteins aberrantly expressed in diverse malignancies, yet whether these proteins contribute to tumor progression remains poorly defined. The CTA Testis-Specific Serine Kinase 6 (TSSK6) is frequently expressed in colorectal cancer (CRC), where it promotes key hallmarks of tumor progression, including anchorage-independent growth, invasion, and in vivo tumor formation. However, the signaling network by which ectopic TSSK6 drives these phenotypes has not been established. Here, we define the signaling pathways regulated by TSSK6 in CRC cells. We demonstrate that TSSK6 promotes focal adhesion kinase (FAK) activation, leading to enhanced STAT3 Ser727 phosphorylation and increased STAT3-dependent transcription. Transcriptomic analyses revealed induction of gene programs enriched for extracellular matrix remodeling, cytoskeletal organization, adhesion, and motility in a STAT3-dependent manner. Disruption of the FAK-STAT3 signaling axis abrogated anchorage-independent growth and invasive outgrowth in a spheroid model. Collectively, these findings demonstrate that aberrant expression of a germline-restricted kinase promotes focal adhesion signaling, which activates STAT3-dependent transcriptional programs that promote anoikis resistance and invasive behavior in CRC.

Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin 6{beta}1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin 6{beta}1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.

Prostate cancer (PCa) is a prevalent male cancer with high survival rates, except in advanced or metastatic stages, for which effective treatments are lacking. Metastatic PCa involves complex mechanisms including loss of tumor suppressor genes and DNA repair molecules, which impacts therapy responses. We have reanalyzed data from a CRISPR/Cas9 genome wide screening previously performed to identify essential regulators of invasive abilities of the metastatic cell line DU145 identifying SYCP3 as a regulator of metastatic invasion. Subsequent analyses of tumor samples demonstrated that SYCP3 expression is frequently upregulated in PCa tumors from patients in advanced stages. Furthermore, SYCP3 genetic depletion significantly reduced the invasive and migratory abilities of DU145 cells and increased their adhesion capacity. Additionally, and due to the implication of SYCP3 on DNA repair processes, we have analyzed the role of SYCP3 on the cellular response to radiotherapy (RT) and found that its depletion induced RT resistance, suggesting a role for SYCP3 in DNA damage response and genomic instability. All these data support a role for SYCP3 in PCa metastasis and provides opportunities for personalized medicine.

Cutaneous melanoma is a highly metastatic cancer in which tumor cell plasticity drives transitions between proliferative and invasive states. The glycolytic regulator PFKFB4 has been implicated in cancer cell motility, but its role in melanoma progression in vivo remains unclear. Using zebrafish larval xenografts, we found that PFKFB4 depletion reduced invasion in MeWo cells without affecting tumor growth, whereas in A375P cells it decreased tumor growth but had no effect on invasion or metastasis. In MeWo, PFKFB4 loss was associated with reduced SNAIL2 expression, while no effect on EMT transcription factors was observed in A375P cells. Rescue experiments indicated that PFKFB4 and SNAIL2 acted independently in regulating Mewo migration in vitro. These findings identify PFKFB4 as a context-dependent regulator of melanoma progression.

Metastasis remains a major cause of morbidity and mortality in men with prostate cancer, and the functional impact of the genetic alterations, alone or in combination, driving metastatic disease remains incompletely understood. The proto-oncogene c-MYC, commonly deregulated in prostate cancer. Transgenic expression of c-MYC is sufficient to drive the progression to prostatic intraepithelial neoplasia and ultimately to moderately differentiated localized primary tumors, however, c-MYC-driven tumors are unable to progress through the metastatic cascade, suggesting that a "second-hit" is necessary in the milieu of aberrant c-MYC-driven signaling. Here, we identified cooperativity between c-MYC and KLF6-SV1, an oncogenic splice variant of the KLF6 gene. Transgenic mice that co-expressed KLF6-SV1 and c-MYC developed progressive and metastatic prostate cancer with a histological and molecular phenotype like human prostate cancer. Silencing c-MYC expression significantly reduced tumor burden in these mice supporting the necessity for c-MYC in tumor maintenance. Unbiased global proteomic analysis of tumors from these mice revealed significantly enriched vimentin, a dedifferentiation and pro-metastatic marker, induced by KLF6-SV1. c-MYC-positive tumors were also significantly enriched for KLF6-SV1 in human prostate cancer specimens. Our findings provide evidence that KLF6-SV1 is an enhancer of c-MYC-driven prostate cancer progression and metastasis, and a correlated genetic event in human prostate cancer with potential translational significance.

The role of autophagy in tumorigenesis and tumor metastasis remains poorly understood. Here we show that inhibition of autophagy stabilizes the transcription factor Twist1 through Sequestosome-1 (SQSTM1, also known as p62) and thus increases cell proliferation, migration, and epithelial-mesenchymal transition (EMT) in tumor development and metastasis. Inhibition of autophagy or p62 overexpression blocks Twist1 protein degradation in the proteasomes, while p62 inhibition enhances it. SQSTM1/p62 interacts with Twist1 via the UBA domain of p62, in a Twist1-ubiquitination-dependent manner. Lysine 175 in Twist1 is critical for Twist1 ubiquitination, degradation, and SQSTM1/p62 interaction. For squamous skin cancer and melanoma cells that express Twist1, SQSTM1/p62 increases tumor growth and metastasis in mice. Together, our results identified Twist1 as a key downstream protein for autophagy and suggest a critical role of the autophagy/p62/Twist1 axis in cancer development and metastasis.

Clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment of a cellular response to hypoxia. Here we show that efferocytic macrophages promote HIF-1 stabilization under normoxic conditions through interaction with phosphorylated STAT3. Inflammatory cytokine gene expression analysis of efferocytic HIF-1-mutant macrophages revealed a reduced expression of the pro-tumorigenic Mif. Furthermore, stabilization of HIF-1 using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in macrophages. Finally, macrophages treated with recombinant MIF protein activated NF-{kappa}B (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by tumor-associated macrophages triggers p-STAT3/HIF-1/MIF signaling to enhance tumor-promoting inflammation in bone, suggesting this axis as a target for metastatic prostate cancer.

Tumor cell intravasation is essential for metastatic dissemination, but its exact mechanism is incompletely understood. We have previously shown that in breast cancer, the direct and stable association of a tumor cell expressing Mena, a Tie2hi/VEGFhi macrophage, and a vascular endothelial cell, creates an intravasation portal, called a "tumor microenvironment of metastasis" (TMEM) doorway, for tumor cell intravasation, leading to dissemination to distant sites. The density of TMEM doorways, also called TMEM doorway score, is a clinically validated prognostic marker of distant metastasis in breast cancer patients. Although we know that tumor cells utilize TMEM doorway-associated transient vascular openings to intravasate, the precise signaling mechanisms involved in TMEM doorway function are only partially understood. Using two mouse models of breast cancer and an in vitro assay of intravasation, we report that CSF-1 secreted by the TMEM doorway tumor cell stimulates local secretion of VEGF-A from the Tie2hi TMEM doorway macrophage, leading to the dissociation of endothelial junctions between TMEM doorway associated endothelial cells, supporting tumor cell intravasation. Acute blockade of CSF-1R signaling decreases macrophage VEGF-A secretion as well as TMEM doorway-associated vascular opening, tumor cell trans-endothelial migration, and dissemination. These new insights into signaling events regulating TMEM doorway function should be explored further as treatment strategies for metastatic disease.

The PEAK family of pseudokinases comprises PEAK1 and PEAK2 as well as the recently-identified PEAK3. PEAK1/2 play fundamental roles in regulating tyrosine kinase signal output and oncogenesis, while PEAK3 remains poorly-characterized. Here, we demonstrate that PEAK3 undergoes homotypic association as well as heterotypic interaction with PEAK1/2. PEAK3 also recruits ASAP1/2, Cbl and PYK2 and the adaptors Grb2 and CrkII, with binding dependent on PEAK3 dimerization. PEAK3 tyrosine phosphorylation on Y24 is also dependent on dimerization as well as Src family kinase activity, and interestingly, is decreased via PTPN12 in response to EGF treatment. Y24 phosphorylation is required for binding of Grb2 and ASAP1. Overexpression of PEAK3 in MDA-MB-231 breast cancer cells enhanced cell elongation and cell motility, while knockdown of endogenous PEAK3 decreased cell migration. In addition, overexpression of PEAK3 in PEAK1/2 compound knock-out MCF-10A breast epithelial cells enhanced acinar growth and invasion in 3D culture, with the latter phenotype dependent on PEAK3 tyrosine phosphorylation and binding of Grb2 and ASAP1. These findings characterize PEAK3 as an integral member of the PEAK family with scaffolding roles that promote cell proliferation, migration and invasion.

Abl family kinases function as proto-oncogenes in various leukemias, and pro-tumor functions have been discovered for Abl kinases in solid tumors as well. However, a growing body of evidence indicates that Abl kinases can function to suppress tumor cell proliferation, motility, and in vivo tumor growth in some settings. To investigate the role of Abl kinases in prostate cancer, we generated Abl-deficient cells in a pre-clinical model of spontaneously metastatic, androgen-indifferent prostate cancer. Loss of Abl family kinase expression resulted in a highly aggressive, metastatic phenotype in vivo that was associated with AKT pathway activation, increased growth on 3D collagen matrix, and enhanced cell motility in vitro. Treatment of Abl kinase-expressing cells with the Abl kinase inhibitor imatinib phenocopied the malignant phenotypes observed in Abl-deficient tumor cells. In addition, inhibiting AKT pathway signaling abolished the increased 3D growth of Abl-deficient cells. Our data reveal that Abl family kinases can function as suppressors of prostate cancer progression and metastasis by restraining AKT signaling, a signaling pathway known to be associated with emergence of metastatic castration-resistant prostate cancer.

Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. While individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a structural complex between receptor tyrosine kinase c-Met and {beta}1 integrin in metastases. Using novel cell culture and in vivo assays, we found that c-Met/{beta}1 complex induction promotes breast cancer intravasation and adhesion to the vessel wall, but does not increase extravasation. These effects may be driven by the ability of the c-Met/{beta}1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/{beta}1 complex induction. A {beta}1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/{beta}1 binding, decreased invasion and mesenchymal gene expression and morphology of breast cancer cells. Bone-seeking breast cancer cells exhibited higher c-Met/{beta}1 complex levels than parental controls and preferentially adhere to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/{beta}1 levels than brain metastases. Thus, the c-Met/{beta}1 complex drives breast cancer cell intravasation and preferential affinity for bone tissue-specific matrix. Pharmacological targeting of the complex may prevent metastases, particularly osseous metastases.

Sensitizing cancer cells to radio- and chemotherapy remains a hot topic in cancer treatment. Here it is identified that Protein Reprimo (RPRM) negatively regulates the levels of ataxia-telangiectasia mutated (ATM) protein kinase, a master regulator of DNA damage response (DDR) in the presence of DNA double-strand breaks (DSBs), resulting in impaired DNA repair efficiency and enhanced cellular sensitivity to genotoxic agents. Mechanistically, although RPRM is primarily located in cytoplasm, it rapidly translocates to nucleus shortly after induced by X-irradiation, interacts with ATM and promotes the nuclear export and proteasomal degradation of ATM. The nuclear translocation of RPRM is associated with its phosphorylation at serine 98, which is mediated by cyclin-dependent kinases 4/6 (CDK4/6). Inhibition of CDK4/6 stabilizes RPRM and promotes its nuclear import, in turn enhances the nuclear export of ATM and the reduction of ATM levels. As a result, RPRM overexpression and its phosphorylation inhibition sensitize cells to genotoxic agents. Moreover, RPRM deficiency significantly increases resistance to radiation-induced damage both in vitro and in vivo. These findings establish a crucial regulatory mechanism in which ATM is negatively modulated by RPRM, suggesting that RPRM may serve as a novel target for both cancer therapy and radiation protection.

Enhanced protein synthesis is a crucial molecular mechanism that allows cancer cells to survive, proliferate, metastasize, and develop resistance to anti-cancer treatments, and often arises as a consequence of increased signaling flux channeled to mRNA-bearing eukaryotic initiation factor 4F (eIF4F). However, the post-translational regulation of eIF4A1, an ATP-dependent RNA helicase and subunit of the eIF4F complex, is still poorly understood. Here, we demonstrate that IBTK, a substrate-binding adaptor of Cullin 3-RING ubiquitin ligase (CRL3) complex, interacts with eIF4A1. The non-degradative ubiquitination of eIF4A1 catalyzed by CRL3IBTK complex promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and tumor cell growth both in vivo and in vitro. Moreover, we show that mTORC1 and S6K1, two key regulators of protein synthesis, directly phosphorylate IBTK to augment eIF4A1 ubiquitination and sustained oncogenic translation. This link between the CRL3IBTK complex and the mTORC1/S6K1 signaling pathway, which is frequently dysregulated in cancer, represents a promising target for anti-cancer therapies. Statement of Significance: Overexpression of IBTK contributes to the process of tumorigenesis by amplifying translation, and represents a promising target for anti-cancer therapies.

Breast cancer remains a leading cause of cancer-related mortality among women, with metastasis being the primary driver of poor prognosis. The ubiquitin-proteasome system (UPS) is a central regulator of protein homeostasis, and its dysregulation is associated with multiple cancers. Within this system, deubiquitinating enzymes (DUBs), which remove ubiquitin moieties from target proteins and thereby modulate their stability and function, have emerged as attractive therapeutic targets. Eukaryotic initiation factor 3 subunit H (EIF3H), a JAMM family DUB, is overexpressed in multiple cancers and implicated in stabilizing oncogenic proteins. Using clinical transcriptomic datasets, we identified EIF3H as significantly upregulated in breast invasive carcinoma, with high expression correlating with poor patient outcomes. Functional assays demonstrated that EIF3H overexpression enhances proliferation, migration, and invasion of breast cancer cells, whereas its knockdown suppresses these traits. Mechanistically, EIF3H physically interacts with and deubiquitinates phosphorylated ERK (pERK), preventing its degradation and sustaining MAPK pathway activation. This represents the first report of pERK as a direct EIF3H substrate, revealing a novel mechanism linking EIF3H to metastatic progression. Moreover, EIF3H-deficient cells display increased sensitivity to chemotherapeutic drugs, suggesting that pharmacological inhibition of EIF3H may simultaneously impair metastasis and improve therapeutic efficacy. Collectively, our findings identify EIF3H as a potential therapeutic target for combating metastatic breast cancer.

Treatment options and diagnostic outlook for men with advanced, therapy resistant prostate cancer (PCa) are extremely poor; this is primarily due to the common lack of durable response to androgen receptor (AR) targeted therapies and phenotypic transdifferentiation into a particularly lethal subtype known as neuroendocrine prostate cancer (NEPC). In this study, we mechanistically determine that SOX2 (a transcription factor originally repressed by AR) physically binds and acts in a concerted manner with FOXA1 (a key AR pioneering cofactor) to regulate a subset of genes which promote cell cycle progression, and lineage plasticity in AR-refractory prostate cancers. Our findings assert the SOX2/FOXA1 interaction as an important mediator of resistance to AR-targeted therapy and a driver of NEPC and lineage plasticity; their coordinated action and downstream signaling offers a potential novel therapeutic opportunity in late-stage PCa.