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.

Spatiotemporal Controllable Sono-Nanovaccines Driven by Free-Field Based Whole-Body Ultrasound for Personalized Cancer Therapy.

Therapeutic cancer vaccines fail to produce satisfactory outcomes against solid tumors since vaccine-induced anti-tumor immunity is significantly hampered by immunosuppression. Generating an in situ cancer vaccine targeting immunological cold tumor microenvironment (TME) appears attractive. Here, a type of free-field based whole-body ultrasound (US)-driven nanovaccines are constructed, named G5-CHC-R, by conjugating the sonosensitizer, Chenghai chlorin (CHC) and the immunomodulator, resiquimod (R848) on top of a super small-sized dendrimeric nanoscaffold. Once entering tumors, R848 can be cleaved from a hypoxia-sensitive linker, thus modifying the TME via converting macrophage phenotypes. The animals bearing orthotopic pancreatic cancer with intestinal metastasis and breast cancer with lung metastasis are treated with G5-CHC-R under a free-field based whole-body US system. Benefit from the deep penetration capacity and highly spatiotemporal selectiveness, G5-CHC-R triggered by US represented a superior alternative for noninvasive irradiation of deep-seated tumors and magnification of local immune responses via driving mass release of tumor antigens and "cold-warm-hot" three-state transformation of TME. In addition to irradiating primary tumors, a robust adaptive anti-tumor immunity is potentiated, leading to successful induction of systemic tumor suppression. The sono-nanovaccines with good biocompatibility posed wide applicability to a broad spectrum of tumors, revealing immeasurable potential for translational research in oncology.

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.

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.

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.

A positive feedback loop involving EGFR/Akt/mTORC1 and IKK/NF-kB regulates head and neck squamous cell carcinoma proliferation.

The overexpression or mutation of epidermal growth factor receptor (EGFR) has been associated with a number of cancers, including head and neck squamous cell carcinoma (HNSCC). Increasing evidence indicates that both the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of Rapamycin (mTOR) and the nuclear factor-kappa B (NF-κB) are constitutively active and contribute to aggressive HNSCC downstream of EGFR. However, whether these two oncogenic signaling pathways exhibit molecular and functional crosstalk in HNSCC is unclear. Our results now reveal that mTORC1, not mTORC2, contributes to NF-κB activation downstream of EGFR/PI3K/Akt signaling. Mechanistically, mTORC1 enhances the inhibitor of nuclear factor kappa-B kinase (IKK) activity to accelerate NF-κB signaling. Concomitantly, activated NF-κB/IKK up-regulates EGFR expression through positive feedback regulation. Blockage of NF-κB/IKK activity by the novel IKKß specific inhibitor, CmpdA, leads to significant inhibition of cell proliferation and induction of apoptosis. CmpdA also sensitizes intrinsic cisplatin-resistant HNSCC cells to cisplatin treatment. Our findings reveal a new mechanism by which EGFR/PI3K/Akt/mTOR signaling promotes head and neck cancer progression and underscores the need for developing a therapeutic strategy for targeting IKK/NF-κB either as a single agent or in combination with cisplatin in head and neck cancer.

IKK phosphorylation of NF-κB at serine 536 contributes to acquired cisplatin resistance in head and neck squamous cell cancer.

Current treatment methods for advanced head and neck squamous cell carcinoma (HNSCC) include surgery, radiation therapy and chemotherapy. For recurrent and metastatic HNSCC, cisplatin is the most common treatment option, but most of patients will eventually develop cisplatin resistance. Therefore, it is imperative to define the mechanisms involved in cisplatin resistance and find novel therapeutic strategies to overcome this deadly disease. In order to determine the role of nuclear factor-kappa B (NF-κB) in contributing to acquired cisplatin resistance in HNSCC, the expression and activity of NF-κB and its upstream kinases, IKKα and IKKß, were evaluated and compared in three pairs of cisplatin sensitive and resistant HNSCC cell lines, including a pair of patient derived HNSCC cell line. The experiments revealed that NF-κB p65 activity was elevated in cisplatin resistant HNSCC cells compared to that in their parent cells. Importantly, the phosphorylation of NF-κB p65 at serine 536 and the phosphorylation of IKKα and IKKß at their activation loops were dramatically elevated in the resistant cell lines. Furthermore, knockdown of NF-κB or overexpression of p65-S536 alanine (p65-S536A) mutant sensitizes resistant cells to cisplatin. Additionally, the novel IKKß inhibitor CmpdA has been shown to consistently block the phosphorylation of NF-κB at serine 536 while also dramatically improving the efficacy of cisplatin in inhibition of cell proliferation and induction of apoptosis in the cisplatin resistant cancer cells. These results indicated that IKK/NF-κB plays a pivotal role in controlling acquired cisplatin resistance and that targeting the IKK/NF-κB signaling pathway may provide a possible therapeutic method to overcome the acquired resistance to cisplatin in HNSCC.

Mitochondria as a critical target of the chemotheraputic agent cisplatin in head and neck cancer.

Cisplatin is among the most important chemotherapeutic agents ever developed. It is a critical component of therapeutic regimens in a broad range of malignancies. However, more than a generation after its clinical introduction, the exact mechanism of cisplatin action on tumor cells is not fully defined. The preponderance of research over the last three decades has focused on cisplatin interactions with nuclear DNA which are felt to lead to apoptotic cell death in sensitive cells. However, recent data have shown that cisplatin may have important direct interactions with mitochondria which can induce apoptosis and may account for a significant portion of the clinical activity associated with this drug. These direct interactions between cisplatin and mitochondria may have critical implications for our understanding of this class of drugs and the development of new therapeutic agents.

Cisplatin preferentially binds mitochondrial DNA and voltage-dependent anion channel protein in the mitochondrial membrane of head and neck squamous cell carcinoma: possible role in apoptosis.

PURPOSE: Cisplatin adducts to nuclear DNA (nDNA) are felt to be the molecular lesions that trigger apoptosis, but the mechanism linking nDNA adduct formation and cell death is unclear. Some literature in the last decade has suggested a possible direct effect of cisplatin on mitochondria independent of nDNA interaction. In this study, we define separately the sequelae of cisplatin interactions with nDNA and with mitochondria in head and neck squamous cell carcinoma (HNSCC) cell lines. EXPERIMENTAL DESIGN: Cisplatin binding to mitochondrial DNA (mtDNA) and proteins was analyzed by atomic absorption spectroscopy and other methods. RESULTS: Following 1 hour of exposure to cisplatin, platinum adducts to mtDNA were 300- to 500-fold more abundant than adducts to nDNA; these differences were not due to differences in rates of adduct repair. Whereas HNSCC cell cytoplasts free of nDNA retained the same dose-dependent cisplatin sensitivity as parental cells, HNSCC rho(0) cells free of mtDNA were 4- to 5-fold more resistant to cisplatin than parental cells. Isolated mitochondria released cytochrome c within minutes of exposure to cisplatin, and ultrastructural analysis of intact HNSCC cells by electron microscopy showed marked mitochondrial disruption after 4 hours of cisplatin treatment, whereas the nucleus and other cellular structures remain intact. The very prompt release of cytochrome c from isolated mitochondria implies that apoptosis does not require alteration in mitochondrial gene transcription. Further, cisplatin binds preferentially to mitochondrial membrane proteins, particularly the voltage-dependent anion channel. CONCLUSIONS: Cisplatin binding to nDNA is not necessary for induction of apoptosis in HNSCC, which can result from direct action of cisplatin on mitochondria.

Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance.

Cisplatin is a potent cytotoxic agent that functions as a bivalent electrophile, forming both interstrand and intrastrand DNA cross-links. Cisplatin-mediated DNA damage results in cell cycle arrest and initiation of apoptotic cell death. Increased cellular glutathione concentrations have been closely correlated with cisplatin resistance but do not reduce the extent of cisplatin-DNA adduct formation. One hypothesis to explain the ability of glutathione to inhibit cisplatin cytotoxicity is that glutathione, through its antioxidant function, plays a role in apoptotic regulatory pathways. We tested this hypothesis using MCF-7 breast cancer cells transfected with the apoptotic inhibitor Bcl-2. Bcl-2 overexpression in MCF-7 cells was associated with a nearly 3-fold increase in cellular glutathione levels and with increased resistance to cell death after cisplatin exposure. Treatment of MCF-7 lines with buthionine sulfoximine, an inhibitor of glutathione synthesis, normalized glutathione levels in Bcl-2 and control transfectants and completely abrogated Bcl-2-mediated cisplatin resistance without affecting Bcl-2 expression. Bcl-2 overexpression and up-regulation of glutathione were not associated with a change in either cisplatin-DNA adduct formation or repair over time. These results suggest that Bcl-2-mediated cisplatin resistance in MCF-7 cells is dependent on up-regulation of glutathione production, which contributes to cell survival by mechanisms independent of cisplatin inactivation or inhibition of DNA adduct formation. A similar dependence on glutathione for Bcl-2-mediated inhibition of cisplatin toxicity was confirmed in a second cell line, the lymphocytic precursor FL5.12. Taken together, these data suggest that apoptotic signaling after genotoxic exposure can be inhibited by the antioxidant activity of glutathione. Inhibition of glutathione synthesis or modulation of glutathione stores in tumors that overexpress Bcl-2 may comprise a novel anticancer strategy.