Differential modulation of PI3K/Akt/mTOR activity by EGFR inhibitors: A rationale for co-targeting EGFR and PI3K in cisplatin-resistant HNSCC.

PURPOSE: To find a new strategy to treat cisplatin-resistant head and neck squamous cell carcinoma (HNSCC), we investigated the effects of EGFR inhibitors on the PI3K/Akt/mTOR pathway and determined the efficacy of EGFR inhibitors in combination with PI3K inhibitors to suppress cell proliferation in cisplatin-resistant-HNSCC. METHODS: The cisplatin-resistant HNSCC cell lines were treated with four FDA approved EGFR inhibitors, which included Gefitinb or Erlotinib alone, or in combination with the pan-PI3K inhibitor, BKM120. Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell proliferation was examined by MTS assay. Apoptosis was analyzed by flow cytometry. RESULTS: Cisplatin-resistant HNSCC cells were also resistant to EGFR inhibitors. However, a combination of EGFR inhibitors with PI3K inhibitor BKM120 dramatically improved the efficacy of EGFR inhibitors to inhibit cell proliferation and induce apoptosis. Furthermore, treatment with EGFR inhibitors differentially affected the phosphorylation of Akt and mTOR, which included partial inhibition, no inhibition, and induction. A combination of EGFR inhibitors and BKM120 completely blocked phosphorylation of EGFR, Akt, and S6K (an mTOR target). CONCLUSION: Our data provided a rationale for EGFR inhibitors in combination with PI3K inhibitors to treat cisplatin-resistant HNSCC.

Adaptive c-Met-PLXDC2 Signaling Axis Mediates Cancer Stem Cell Plasticity to Confer Radioresistance-associated Aggressiveness in Head and Neck Cancer.

Radiotherapy plays an essential role in the treatment of head and neck squamous cell carcinoma (HNSCC), yet radioresistance remains a major barrier to therapeutic efficacy. A better understanding of the predominant pathways determining radiotherapy response could help develop mechanism-informed therapies to improve cancer management. Here we report that radioresistant HNSCC cells exhibit increased tumor aggressiveness. Using unbiased proteome profiler antibody arrays, we identify that upregulation of c-Met phosphorylation is one of the critical mechanisms for radioresistance in HNSCC cells. We further uncover that radioresistance-associated HNSCC aggressiveness is effectively exacerbated by c-Met but is suppressed by its genetic knockdown and pharmacologic inactivation. Mechanistically, the resulting upregulation of c-Met promotes elevated expression of plexin domain containing 2 (PLXDC2) through activating ERK1/2-ELK1 signaling, which in turn modulates cancer cell plasticity by epithelial-mesenchymal transition (EMT) induction and enrichment of the cancer stem cell (CSC) subpopulation, leading to resistance of HNSCC cells to radiotherapy. Depletion of PLXDC2 overcomes c-Met-mediated radioresistance through reversing the EMT progress and blunting the self-renewal capacity of CSCs. Therapeutically, the addition of SU11274, a selective and potent c-Met inhibitor, to radiation induces tumor shrinkage and limits tumor metastasis to lymph nodes in an orthotopic mouse model. Collectively, these significant findings not only demonstrate a novel mechanism underpinning radioresistance-associated aggressiveness but also provide a possible therapeutic strategy to target radioresistance in patients with HNSCC. Significance: This work provides novel insights into c-Met-PLXDC2 signaling in radioresistance-associated aggressiveness and suggests a new mechanism-informed therapeutic strategy to overcome failure of radiotherapy in patients with HNSCC.

Co-Targeting ErbB Receptors and the PI3K/AKT Axis in Androgen-Independent Taxane-Sensitive and Taxane-Resistant Human Prostate Cancer Cells.

Using two representative models of androgen-independent prostate cancer (PCa), PC3 and DU145, and their respective paclitaxel- and docetaxel-resistant derivatives, we explored the anti-tumor activity of targeting the ErbB receptors and AKT using small-molecule kinase inhibitors. These cells manifest varying degrees of neuroendocrine differentiation characteristics and differ in their expression of functional PTEN. Although the specific downstream signaling events post the ErbB receptor and AKT co-targeting varied between the PC3- and DU145-lineage cells, synergistic anti-proliferative and enhanced pro-apoptotic responses occurred across the wild-type and the taxane-resistant cells, independent of their basal AKT activation state, their degree of paclitaxel- or docetaxel-resistance, or whether this resistance was mediated by the ATP Binding Cassette transport proteins. Dual targeting also led to enhanced anti-tumor responses in vivo, although there was pharmacodynamic discordance between the PCa cells in culture versus the tumor xenografts in terms of the relative activation and inhibition states of AKT and ERK under basal conditions and upon AKT and/or ErbB targeting. The consistent inhibition, particularly of AKT, occurred both in vitro and in vivo, independent of the underlying PTEN status. Thus, co-targeting AKT with ErbB, and possibly other partners, may be a useful strategy to explore further for potential therapeutic effect in advanced PCa.

Simultaneously targeting ErbB family kinases and PI3K in HPV-positive head and neck squamous cell carcinoma.

OBJECTIVES: To identify the most effective PI3K and EGFR inhibitors in HPV-positive head and neck squamous cell carcinoma (HNSCC) and investigate the efficacy of a combination of an ErbB family kinase inhibitor and a PI3K inhibitor to inhibit cell proliferation of HPV-positive HNSCC. MATERIALS AND METHOD: HPV-positive HNSCC cell lines were treated with the FDA approved ErbB kinase inhibitor, Afatinib or FDA-approved PI3K inhibitor, Copanlisib, alone or in combination, and phosphorylation and total protein levels of cells were assessed by Western blot analysis.Cell proliferation and apoptosis were examined by MTS assay, flow cytometry, and Western blots, respectively. RESULTS: Copanlisib more effectively inhibited cell proliferation in comparison to other PI3K inhibitors tested. HPV-positive HNSCC cells differentially responded to cisplatin, Afatinib, or Copanlisib. The combination of Afatinib and Copanlisib more effectively suppressed cell proliferation and induced apoptosis compared to either treatment alone. Mechanistically, the combination of Afatinib and Copanlisib completely blocked phosphorylation of EGFR, HER2, HER3, and Akt as well as significantly decreased the HPV E7 expression compared to either treatment alone. CONCLUSION: Afatinib and Copanlisib more effectively suppress cell proliferation and survival of HPV-positive HNSCC in comparison to either treatment alone.

Targeting Wee1 kinase to suppress proliferation and survival of cisplatin-resistant head and neck squamous cell carcinoma.

PURPOSE: We investigated the role of Wee1 kinase in cisplatin-resistant head and neck squamous cell carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines and determined the efficacy of either Wee1 inhibitor, AZD1775 alone, or in combination with cisplatin, on cisplatin-resistant HNSCC inhibition. METHODS: Phosphorylation and total protein levels of cells were assessed by Western blot analysis. Cell viability and apoptosis were examined by MTS assay and flow cytometry, respectively. RESULTS: Wee1 kinase protein expression levels in five cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin-sensitive partners. Importantly, Wee1 knockdown inhibited cell proliferation and re-sensitized cells to cisplatin treatment. Interestingly, previous studies have also shown that Wee1 inhibitor AZD1775 synergizes with cisplatin to suppress cell proliferation of cisplatin-sensitive HNSCC. We found that AZD1775 inhibited both cisplatin-sensitive and resistant HNSCC with similar IC50 values, which suggested that AZD1775 could overcome cisplatin resistance in cisplatin-resistant HNSCC. Mechanistically, AZD1775 and cisplatin cooperatively induced DNA damage and apoptosis. CONCLUSION: Wee1 inhibitor, AZD1775, and cisplatin coordinately suppressed proliferation and survival of HNSCC.

Inhibition of IKKß/NF-κB signaling pathway to improve Dasatinib efficacy in suppression of cisplatin-resistant head and neck squamous cell carcinoma.

Proto-oncogene tyrosine-protein kinase Src plays an important role in Head and Neck Squamous Cell Carcinoma (HNSCC). However, the FDA-approved SRC inhibitor Dasatinib shows very limited efficacy in HNSCC clinical trials, even though Dasatinib can completely inhibit SRC in the laboratory setting. These results suggest that SRC inhibition can cause compensatory up-regulation and/or activation of other survival pathways, which suggests that co-targeting of SRC and the potential signaling pathways may improve the Dasatinib efficacy. In this study, we investigated the role of IKKß/NF-κB in regulation of the sensitivity of cisplatin-resistant HNSCC to Dasatinib. Additionally, we wished to determine whether inhibition of the IKKß/NF-κB signaling pathway could enhance Dasatinib efficacy to inhibit cisplatin-resistant HNSCC without the use of cisplatin. Previous studies have shown that ETS-1 is a crucial SRC effector protein that regulates cancer cell proliferation, anti-apoptosis, and metastasis. We found that SRC kinase inhibition by Dasatinib decreased ETS-1 expression but caused elevation of IKKß/NF-κB signaling in multiple cisplatin-resistant HNSCC. Interestingly, inhibition of IKKß/NF-κB by CmpdA (Bay65-1942), a recently identified IKKß inhibitor, also led to a decrease in ETS-1 levels. Moreover, the knockdown of IKK, but not NF-κB, dramatically decreased ETS-1 expression. In addition, IKKß and ETS-1 interacted in cisplatin-resistant HNSCC. These data demonstrated cross-talk between SRC and IKK to regulate NF-κB and ETS-1. Furthermore, we found that simultaneous inhibition of SRC and IKKß through a Dasatinib and CmpdA combination synergistically inhibited NF-κB activation and ETS-1expression, suppressed cell proliferation, and induced apoptosis. Taken together, our data indicate that SRC and IKKß play crucial roles in cisplatin-resistant HNSCCC and co-targeting SRC and IKKß could be an effective strategy to treat cisplatin-resistant HNSCC.

Concurrent inhibition of ErbB family and MEK/ERK kinases to suppress non-small cell lung cancer proliferation.

Lung cancer ranks as the most common cancer and leading cause of cancer-related deaths worldwide. Of all lung cancer types, non-small cell lung cancer (NSCLC) accounts for 85 percent of all cases. The high mortality of NSCLC occurs mainly because of poor prognosis in patients with recurrent and metastatic cancer. Cisplatin-containing chemotherapy is the first option to treat recurrent and metastatic NSCLC. Additionally, targeted therapy plays an important role to prolong life in patients. Currently, EGFR inhibitors are the most important targeted anti-cancer drugs for patients with EGFR mutations in the clinical setting. Another important kinase inhibitor for targeted therapy is the MEK inhibitor, Trametinib, which is often used for patients with BRAF mutation or MEK/ERK activation in the tumors. In this study, we determined whether a combination of the pan-ErbB kinase inhibitor, Afatinib, and MEK inhibitor, Trametinib, could more effectively inhibit NSCLC cell proliferation when compared to either single treatment. We found that Afatinib inhibited phosphorylation of EGFR, HER2, HER3, and HER4, as well as Akt, whereas it elevated ERK phosphorylation. Conversely, Trametinib treatment led to ERK inhibition, but induced Akt phosphorylation. However, the combination of Afatinib and Trametinib inhibited all of the above-mentioned signaling pathways and synergistically suppressed cell proliferation. Our data indicate that co-targeting of ErbB family and MEK/ERK pathways through a combination of Afatinib and Trametinib could be a potential effective strategy to treat NSCLC.

Suppression of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma through IKKß inhibition.

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKß in regulation of migration, invasion, and metastasis of cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We showed that cisplatin-resistant HNSCC cells have a stronger ability to migrate and invade, as well as display higher IKKß/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKß, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKß inhibitor, CmpdA, also inhibited cell migration and invasion. Previous studies have already shown that N-Cadherin, an epithelial-mesenchymal transition (EMT) marker, and IL-6, a pro-inflammatory cytokine, play important roles in regulation of HNSCC migration, invasion, and metastasis. We found that cisplatin-resistant HNSCC expressed higher levels of N-Cadherin and IL-6, which were significantly inhibited by CmpdA. More importantly, we showed that CmpdA treatment dramatically abated cisplatin-resistant HNSCC cell metastasis to lungs in a mouse model. Our data demonstrated the crucial role of IKKß in control of migration, invasion, and metastasis, and implicated that targeting IKKß may be a potential therapy for cisplatin-resistant metastatic HNSCC.

Conditional Reprogramming for Patient-Derived Cancer Models and Next-Generation Living Biobanks.

Traditional cancer models including cell lines and animal models have limited applications in both basic and clinical cancer research. Genomics-based precision oncology only help 2-20% patients with solid cancer. Functional diagnostics and patient-derived cancer models are needed for precision cancer biology. In this review, we will summarize applications of conditional cell reprogramming (CR) in cancer research and next generation living biobanks (NGLB). Together with organoids, CR has been cited in two NCI (National Cancer Institute, USA) programs (PDMR: patient-derived cancer model repository; HCMI: human cancer model initiatives. HCMI will be distributed through ATCC). Briefly, the CR method is a simple co-culture technology with a Rho kinase inhibitor, Y-27632, in combination with fibroblast feeder cells, which allows us to rapidly expand both normal and malignant epithelial cells from diverse anatomic sites and mammalian species and does not require transfection with exogenous viral or cellular genes. Establishment of CR cells from both normal and tumor tissue is highly efficient. The robust nature of the technique is exemplified by the ability to produce 2 × 106 cells in five days from a core biopsy of tumor tissue. Normal CR cell cultures retain a normal karyotype and differentiation potential and CR cells derived from tumors retain their tumorigenic phenotype. CR also allows us to enrich cancer cells from urine (for bladder cancer), blood (for prostate cancer), and pleural effusion (for non-small cell lung carcinoma). The ability to produce inexhaustible cell populations using CR technology from small biopsies and cryopreserved specimens has the potential to transform biobanking repositories (NGLB: next-generation living biobank) and current pathology practice by enabling genetic, biochemical, metabolomic, proteomic, and biological assays, including chemosensitivity testing as a functional diagnostics tool for precision cancer medicine. We discussed analyses of patient-derived matched normal and tumor models using a case with tongue squamous cell carcinoma as an example. Last, we summarized applications in cancer research, disease modeling, drug discovery, and regenerative medicine of CR-based NGLB.

Inhibition of cisplatin-resistant head and neck squamous cell carcinoma by combination of Afatinib with PD0325901, a MEK inhibitor.

ErbB family members that contain EGFR, HER2, HER3 and HER4 play important roles in many cancer types, including head and neck; however, inhibition of these receptors by small molecule kinase inhibitors showed limited results due to compensatory up-regulation of some key survival signaling pathways. Here, we explore the effectiveness of Afatinib, an irreversible inhibitor of EGFR, HER2, and HER4, in combination with the MEK inhibitor PD0325901 to inhibit cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). We treated two cisplatin-resistant HNSCC cell lines, UMSCC74B and O28, with Afatinib, PD0325901, or a combination, and measured signaling pathways, cell proliferation, and survival. We found that Afatinib blocked Akt/mTOR activity and phosphorylation of EGFR, HER2 and HER3, but up-regulated MEK/ERK signaling. Interestingly, MEK inhibitor PD0325901 blocked ERK phosphorylation, but elevated phosphorylation of Akt and mTOR pathways. Similarly, Afatinib and PD0325901 inhibited all these pathways and synergistically suppressed cell proliferation and survival. Our data demonstrate that Afatinib in combination with MEK inhibitors could provide a potential novel therapy for cisplatin-resistant head and neck squamous cell cancer.

Regulation of cisplatin-resistant head and neck squamous cell carcinoma by the SRC/ETS-1 signaling pathway.

BACKGROUND: We investigated the role of the ETS-1 transcription factor in Head and Neck Squamous Cell Carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines. METHODS: We examined its molecular link with SRC and MEK/ERK pathways and determined the efficacy of either MEK/ERK inhibitor PD0325901 or SRC inhibitor Dasatinib on cisplatin-resistant HNSCC inhibition. RESULTS: We found that ETS-1 protein expression levels in a majority of cisplatin-resistant HNSCC cell types were higher than those in their parental cisplatin sensitive partners. High ETS-1 expression was also found in patient-derived, cisplatin-resistant HNSCC cells. While ETS-1 knockdown inhibited cell proliferation, migration, and invasion, it could still re-sensitize cells to cisplatin treatment. Interestingly, previous studies have shown that MER/ERK pathways could regulate ETS-1 through its phosphorylation at threonine 38 (T38). Although almost all cisplatin-resistant HNSCC cells we tested showed higher ETS-1 phosphorylation levels at T38, we found that inhibition of MEK/ERK pathways with the MEK inhibitor PD0325901 did not block this phosphorylation. In addition, treatment of cisplatin-resistant HNSCC cells with the MEK inhibitor completely blocked ERK phosphorylation but did not re-sensitize cells to cisplatin treatment. Furthermore, we found that, consistent with ETS-1 increase, SRC phosphorylation dramatically increased in cisplatin-resistant HNSCC, and treatment of cells with the SRC inhibitor, Dasatinib, blocked SRC phosphorylation and decreased ETS-1 expression. Importantly, we showed that Dasatinib, as a single agent, significantly suppressed cell proliferation, migration, and invasion, in addition to survival. CONCLUSIONS: Our results demonstrate that the SRC/ETS-1 pathway plays a crucial role and could be a key therapeutic target in cisplatin-resistant HNSCC treatment.

Co-targeting EGFR and IKKß/NF-κB signalling pathways in head and neck squamous cell carcinoma: a potential novel therapy for head and neck squamous cell cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) plays an important role in head and neck squamous cell carcinoma (HNSCC) proliferation and therapy resistance, but the efficacy of targeting of EGFR for therapy has been limited. Here, we explore the molecular link between EGFR and inhibitor of κB kinase ß/nuclear factor-κB (IKKß/NF-κB) signalling pathways in the regulation of HNSCC EGFR inhibitor resistance. METHODS: We performed in vitro experiments in eight human HNSCC cell lines and a patient-derived HNSCC cell line as well as in vivo xenografts in a human HNSCC cell line. RESULTS: We found that treatment of all HNSCC cells with Gefitinib and Erlotinib, two Food Drug Administration-approved EGFR inhibitors, blocked the activity of Akt/mammalian target of the rapamycin (mTOR) and extracellular signal-regulated kinase, two crucial downstream effectors of EGFR, but up-regulated IKKß/NF-κB signalling. In addition, induction of IKKß/NF-κB by EGFR inhibitors required HER2 and HER3 expression. In keeping with these, IKKß inhibitor CmpdA synergistically enhanced the efficacy of EGFR inhibitors to further inhibit in vitro HNSCC cell growth. Importantly, we demonstrated that the combination of Gefitinib with CmpdA inhibited xenograft tumour formation. CONCLUSION: Our data demonstrated that co-targeting EGFR and IKKß with Gefitinib and IKKß inhibitors could provide a potential novel therapy for head and neck squamous cell cancer.

Decreased TRPM7 inhibits activities and induces apoptosis of bladder cancer cells via ERK1/2 pathway.

Transient receptor potential melastatin 7 (TRPM7) functions as a Mg2+/Ca2+-permeable channel fused with a kinase domain and regulates various physical processes and diseases. However, its effects on pathogenesis of human bladder cancer (BCa) has not been clarified yet. Our microarray analysis has suggested that calcium signaling pathway is connected with bladder cancer via MAPK pathway. Therefore, we aim to investigate the mechanism of TRPM7 in BCa tumorigenesis by using BCa tissues compared with normal bladder epithelium tissues, as well as using distinct BCa cell lines (EJ, 5637 and T24). We observed increased TRPM7 expression and dysregulation of proteins involved in Epithelial-Mesenchymal Transition (EMT) in BCa tissues. Moreover, knockdown of TRPM7 in BCa cells reversed the EMT status, accompanied by increase of reactive oxygen species (ROS). Furthermore, TRPM7 deficiency could inhibit BCa cell proliferation, migration and invasion, as well as induce p-ERK1/2 and suppress PI3K/AKT at the protein level. Downregulation of TRPM7 promoted cell cycle arrest at G0/G1 phase and apoptosis in vitro, which could be recovered by pre-treatment with U0126 to deactivate ERK1/2, suggesting a close correlation between TRPM7 and the MAPK signaling pathway. Furthermore, a NOD/SCID mouse model transplanted using the BCa cells was established, revealing delayed tumor growth by reduced protein activity and mRNA transcription of TRPM7 in vivo. Our results suggested TRPM7 might be essential for BCa tumorigenesis by interfering BCa cell proliferation, motility and apoptosis.

Oncogenic EGFR signaling activates an mTORC2-NF-κB pathway that promotes chemotherapy resistance.

UNLABELLED: Although it is known that mTOR complex 2 (mTORC2) functions upstream of Akt, the role of this protein kinase complex in cancer is not well understood. Through an integrated analysis of cell lines, in vivo models, and clinical samples, we demonstrate that mTORC2 is frequently activated in glioblastoma (GBM), the most common malignant primary brain tumor of adults. We show that the common activating epidermal growth factor receptor (EGFR) mutation (EGFRvIII) stimulates mTORC2 kinase activity, which is partially suppressed by PTEN. mTORC2 signaling promotes GBM growth and survival and activates NF-κB. Importantly, this mTORC2-NF-κB pathway renders GBM cells and tumors resistant to chemotherapy in a manner independent of Akt. These results highlight the critical role of mTORC2 in the pathogenesis of GBM, including through the activation of NF-κB downstream of mutant EGFR, leading to a previously unrecognized function in cancer chemotherapy resistance. These findings suggest that therapeutic strategies targeting mTORC2, alone or in combination with chemotherapy, will be effective in the treatment of cancer. SIGNIFICANCE: This study demonstrates that EGFRvIII-activated mTORC2 signaling promotes GBM proliferation, survival, and chemotherapy resistance through Akt-independent activation of NF-κB. These results highlight the role of mTORC2 as an integrator of two canonical signaling networks that are commonly altered in cancer, EGFR/phosphoinositide-3 kinase (PI3K) and NF-κB. These results also validate the importance of mTORC2 as a cancer target and provide new insights into its role in mediating chemotherapy resistance, suggesting new treatment strategies.