Repression of AXL expression by AP-1/JNK blockage overcomes resistance to PI3Ka therapy.
ABSTRACT: AXL overexpression is a common resistance mechanism to anti-cancer therapies, including the resistance to BYL719 (Alpelisib) - the p110α isoform specific inhibitor of phosphoinositide 3-kinase (PI3K) - in esophagus and head and neck squamous cell carcinoma (ESCC, HNSCC respectively). However, the mechanisms underlying AXL overexpression in resistance to BYL719 remain elusive. Here we demonstrated that the AP-1 transcription factors, c-JUN and c-FOS, regulate AXL overexpression in HNSCC and ESCC. The expression of AXL was correlated with that of c-JUN both in HNSCC patients and in HNSCC and ESCC cell lines. Silencing of c-JUN and c-FOS expression in tumor cells downregulated AXL expression and enhanced the sensitivity of human papilloma virus positive (HPVPos) and negative (HPVNeg) tumor cells to BYL719 in vitro. Blocking of the c-JUN N-terminal kinase (JNK) using SP600125 in combination with BYL719 showed a synergistic anti-proliferative effect in vitro, which was accompanied by AXL downregulation and potent inhibition of the mTOR pathway. In vivo, the BYL719-SP600125 drug combination led to the arrest of tumor growth in cell line-derived and patient-derived xenograft models, and in syngeneic head and neck murine cancer models. Collectively, our data suggests that JNK inhibition in combination with anti-PI3K therapy is a new therapeutic strategy that should be tested in HPVPos and HPVNeg HNSCC and ESCC patients.
Project description:FRA1 (Fos-like antigen 1) is highly expressed in many epithelial cancers including squamous cell carcinoma of the skin (cSCC) and head and neck (HNSCC). However, the functional importance and the mechanisms mediating FRA1 function in these cancers are not fully understood. Here, we demonstrate that FRA1 gene silencing in HNSCC and cSCC cells resulted in two consequences - impaired cell proliferation and migration. FRA1 regulation of cell growth was distinct from that of c-Jun, a prominent Jun group AP-1 factor. While c-Jun was required for the expression of the G1/S phase cell cycle promoter CDK4, FRA1 was essential for AKT activation and AKT-dependent expression of CyclinB1, a molecule required for G2-M progression. Exogenous expression of a constitutively active form of AKT rescued cancer cell growth defect caused by FRA1-loss. Additionally, FRA1 knockdown markedly slowed cell adhesion and migration, and conversely expression of an active FRA1 mutant (FRA1DD) expedited these processes in a JNK/c-Jun-dependent manner. Through protein and ChIP-PCR analyses, we identified KIND1, a cytoskeletal regulator of the cell adhesion molecule ?1-integrin, as a novel FRA1 transcriptional target. Restoring KIND1 expression rescued migratory defects induced by FRA1 loss. In agreement with these in vitro data, HNSCC cells with FRA1 loss displayed markedly reduced rates of subcutaneous tumor growth and pulmonary metastasis. Together, these results indicate that FRA1 promotes cancer growth through AKT, and enhances cancer cell migration through JNK/c-Jun, pinpointing FRA1 as a key integrator of JNK and AKT signaling pathways and a potential therapeutic target for cSCC and HNSCC.
Project description:Head and neck squamous cell carcinoma (HNSCC) remains a clinical challenge and identification of novel therapeutic targets is necessary. The receptor tyrosine kinase AXL has been implicated in several tumor entities and a selective AXL small molecule inhibitor (BGB324) is currently being tested in clinical trials for patients suffering from non-small cell lung cancer or acute myeloid leukemia. Our study investigates AXL expression during HNSCC progression and its use as a potential therapeutic target in HNSCC. AXL protein expression was determined in a HNSCC cohort (n = 364) using immunohistochemical staining. For functional validation, AXL was either overexpressed or inhibited with BGB324 in HNSCC cell lines to assess proliferation, migration and invasion. We found AXL protein expression increasing during tumor progression with highest expression levels in recurrent tumors. In HNSCC cell lines in vitro, AXL overexpression increased migration as well as invasion. Both properties could be reduced through treatment with BGB324. In contrast, proliferation was neither affected by AXL overexpression nor by inhibition with BGB324. Our patient-derived data and in vitro results show that, in HNSCC, AXL is important for the progression to more advanced tumor stages. Moreover, they suggest that AXL could be a target for precision medicine approaches in this dismal tumor entity.
Project description:Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5'-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.
Project description:Receptor tyrosine kinases (RTKs) and Yes-associated protein (YAP) are critical driving factors in tumors. Currently, the regulation of RTKs in the Hippo-YAP pathway has been recognized as an important issue. However, the relationship between AXL, one of the RTKs, and YAP in head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, the crosstalk between AXL and YAP was thoroughly investigated in vitro and in vivo. We determined that there was a positive correlation between AXL and YAP in the HNSCC tissue samples and the Cancer Genome Atlas (TCGA) dataset, and high co-expression was associated with poor prognosis. Inhibiting YAP decreased AXL expression in HNSCC cells, while YAP overexpression increased AXL. Moreover, ectopic expression of AXL reversed tumor suppressor phenotypes mediated by YAP silencing. This reversal effect was also confirmed in vivo. In addition, AXL overexpression and Gas6, a ligand of AXL, stimulated YAP dephosphorylation, nuclear translocation, and target gene transcription. AXL inhibition decreased YAP dephosphorylation and nuclear translocation. Mechanistically, Gas6 induced a competitive binding to phosphorylated signal transducers and activators of transcription 3 (STAT3) with large tumor suppressor kinase 1 (LATS1) and inhibited the Hippo pathway. This study revealed a novel non-transcriptional effect of STAT3 in Gas6/AXL-induced YAP activity, suggesting that STAT3 acted as a critical "molecular switch" during the mutual promotion between AXL and YAP, which might be a promising therapeutic target in HNSCC.
Project description:BACKGROUND:Aberrant activation of the phosphatidylinositol 3-kinase (PI3K) pathway is common in many malignancies, including head and neck squamous cell carcinoma (HNSCC). Despite pre-clinical and clinical studies, outcomes from targeting the PI3K pathway have been underwhelming and the development of drug resistance poses a significant barrier to patient treatment. In the present study, we examined mechanisms of acquired resistance to the PI3K? inhibitor alpelisib (formerly BYL719) in HNSCC cell lines and patient-derived xenografts (PDXs). METHODS:Five unique PDX mouse models and three HNSCC cell lines were used. All cell lines and xenografts underwent genomic characterization prior to study. Serial drug treatment was conducted in vitro and in vivo to develop multiple, clinically-significant models of resistance to alpelisib. We then used reverse phase protein arrays (RPPAs) to profile the expression of proteins in parental and drug-resistant models. Top hits were validated by immunoblotting and immunohistochemistry. Flow cytometric analysis and RNA interference studies were then used to interrogate the molecular mechanisms underlying acquired drug resistance. RESULTS:Prolonged treatment with alpelisib led to upregulation of TAM family receptor tyrosine kinases TYRO3 and AXL. Importantly, a significant shift in expression of both TYRO3 and AXL to the cell surface was detected in drug-resistant cells. Targeted knockdown of TYRO3 and AXL effectively re-sensitized resistant cells to PI3K? inhibition. In vivo, resistance to alpelisib emerged following 20-35?days of treatment in all five PDX models. Elevated TYRO3 expression was detected in drug-resistant PDX tissues. Downstream of TYRO3 and AXL, we identified activation of intracellular MAPK signalling. Inhibition of MAPK signalling also re-sensitized drug-resistant cells to alpelisib. CONCLUSIONS:We have identified TYRO3 and AXL receptors to be key mediators of resistance to alpelisib, both in vitro and in vivo. Our findings suggest that pan-TAM inhibition is a promising avenue for combinatorial or second-line therapy alongside PI3K? inhibition. These findings advance our understanding of the role TAM receptors play in modulating the response of HNSCC to PI3K? inhibition and suggest a means to prevent, or at least delay, resistance to PI3K? inhibition in order to improve outcomes for HNSCC patients.
Project description:Resistance to antitumor immunity can be promoted by the oncogenic pathways operational in human cancers, including the epidermal growth factor receptor (EGFR) pathway. Here we studied if and how EGFR downstream signaling in head and neck squamous cell carcinoma (HNSCC) can affect the attraction of immune cells. HPV-negative and HPV-positive HNSCC cell lines were analyzed in vitro for CCL2, CCL5, CXCL9, CXCL10, IL-6 and IL-1? expression and the attraction of T cells under different conditions, including cetuximab treatment and stimulation with IFN? and TNF? using qPCR, ELISA and migration experiments. Biochemical analyses with chemical inhibitors and siRNA transfection were used to pinpoint the underlying mechanisms. Stimulation of HNSCC cells with IFN? and TNF? triggered the production of T-cell attracting chemokines and required c-RAF activation. Blocking of the EGFR with cetuximab during this stimulation increased chemokine production in vitro, and augmented the attraction of T cells. Mechanistically, cetuximab decreased the phosphorylation of MEK1, ERK1/2, AKT, mTOR, JNK, p38 and ERK5. Chemical inhibition of EGFR signaling showed a consistent and pronounced chemokine production with MEK1/2 inhibitor PD98059 and JNK inhibitor SP600125, but not with inhibitors of p38, PI3K or mTOR. Combination treatment with cetuximab and a MEK1/2 or JNK inhibitor induced the highest chemokine expression. In conclusion, overexpression of EGFR results in the activation of multiple downstream signaling pathways that act simultaneously to suppress type 1 cytokine stimulated production of chemokines required to amplify the attraction of T cells.
Project description:BEX2 has been suggested to promote the tumor growth in breast cancer and glioblastoma, while inhibit the proliferation of glioma cells. Thus, the role of BEX2 in tumor was still in debate. Additionally, the biological functions of BEX2 in colorectal cancer (CRC) have not yet been clarified. Here, we reported that BEX2 was overexpressed in advanced CRC from both the GSE14333 database and fresh CRC tissue specimens, and positively correlated with clinical staging. Knockdown of BEX2 significantly decreased the in vitro proliferation of SW620 colorectal cancer cells, suppressed subcutaneous xenograft growth and enhanced the survival of mice with cecal tumors. These effects were mainly mediated by the JNK/c-Jun pathway. Knockdown of BEX2 inhibited JNK/c-Jun phosphorylation, while BEX2 overexpression activated JNK/c-Jun phosphorylation. Moreover, the administration of the JNK-specific inhibitor SP600125 to SW620 with BEX2 overexpression abolished the effect of BEX2 on SW620 cell proliferation. This study reveals that BEX2 promotes colorectal cancer cell proliferation via the JNK/c-Jun pathway, suggesting BEX2 as a potential candidate target for the treatment of CRC.
Project description:We hypothesized that targeting key points in the ischemic cascade with combined neuroglobin (Ngb) overexpression and c-jun N-terminal kinase (JNK) inhibition (SP600125) would offer greater neuroprotection than single treatment after in vitro hypoxia/reoxygenation and in a randomized, blinded in vivo experimental stroke study using a clinically relevant rat strain. Male spontaneously hypertensive stroke-prone rats underwent transient middle cerebral artery occlusion (tMCAO) and were divided into the following groups: tMCAO; tMCAO+control GFP-expressing canine adenovirus-2, CAVGFP; tMCAO+Ngb-expressing CAV-2, CAVNgb; tMCAO+SP600125; tMCAO+CAVNgb+SP600125; or sham procedure. Rats were assessed till day 14 for neurologic outcome before infarct determination. In vitro, combined lentivirus-mediated Ngb overexpression+SP600125 significantly reduced oxidative stress and apoptosis compared with single treatment(s) after hypoxia/reoxygenation in B50 cells. In vivo, infarct volume was significantly reduced by CAVNgb, SP600125, and further by CAVNgb+SP600125. The number of Ngb-positive cells in the peri-infarct cortex and striatum was significantly increased 14 days after tMCAO in animals receiving CAVNgb. Neurologic outcome, measured using a 32-point neurologic score, significantly improved with CAVNgb+SP600125 compared with single treatments at 14 days after tMCAO. Combined Ngb overexpression with JNK inhibition reduced hypoxia/reoxygenation-induced oxidative stress and apoptosis in cultured neurons and reduced infarct and improved neurologic outcome more than single therapy after in vivo experimental stroke in hypertensive rats.
Project description:We previously reported that IGF binding protein-3 (IGFBP-3), a major IGF-binding protein in human serum, regulates angiogenic activities of human head and neck squamous cell carcinoma (HNSCC) cells and human umbilical vein endothelial cells (HUVECs) through IGF-dependent and IGF-independent mechanisms. However, the role of IGFBP-3 in cell adhesion is largely unknown. We demonstrate here that IGFBP-3 inhibits the adhesion of HNSCC cells and HUVECs to the extracellular matrix (ECM). IGFBP-3 reduced transcription of a variety of integrins, especially integrin ?4, and suppressed phosphorylation of focal adhesion kinase (FAK) and Src in these cells through both IGF-dependent and IGF-independent pathways. IGFBP-3 was found to suppress the transcription of c-fos and c-jun and the activity of AP1 transcription factor. The regulatory effect of IGFBP-3 on integrin ?4 transcription was attenuated by blocking c-jun and c-fos gene expression via siRNA transfection. Taken together, our data show that IGFBP-3 has IGF-dependent and -independent inhibitory effects on intracellular adhesion signaling in HNSCC and HUVECs through its ability to block c-jun and c-fos transcription and thus AP-1-mediated integrin ?4 transcription. Collectively, our data suggest that IGFPB-3 may be an effective cancer therapeutic agent by blocking integrin-mediated adhesive activity of tumor and vascular endothelial cells.