Cisplatin-resistant Head And Neck Squamous Cell Carcinoma Research Articles

Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma

ABSTRACT Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

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

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. 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. 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). Our data provided a rationale for EGFR inhibitors in combination with PI3K inhibitors to treat cisplatin-resistant HNSCC.

Pharmacological inhibition of MYC to mitigate chemoresistance in preclinical models of squamous cell carcinoma.

Rationale: Cisplatin-based chemotherapy is the first-line treatment for late-stage head and neck squamous cell carcinoma (HNSCC). However, resistance to cisplatin has become a major obstacle for effective therapy. Cancer stem cells (CSCs) are critical for tumor initiation, growth, metastasis, and chemoresistance. How to effectively eliminate CSCs and overcome chemoresistance remains a key challenge. Herein, we confirmed that MYC plays critical roles in chemoresistance, and explored targeting MYC to overcome cisplatin resistance in preclinical models. Methods: The roles of MYC in HNSCC cisplatin resistance and cancer stemness were tested in vitro and in vivo. The combined therapeutic efficiency of MYC targeting using the small molecule MYC inhibitor MYCi975 and cisplatin was assessed in a 4‑nitroquinoline 1-oxide-induced model and in a patient-derived xenograft model. Results: MYC was highly-expressed in cisplatin-resistant HNSCC. Targeting MYC using MYCi975 eliminated CSCs, prevented metastasis, and overcame cisplatin resistance. MYCi975 also induced tumor cell-intrinsic immune responses, and promoted CD8+ T cell infiltration. Mechanistically, MYCi975 induced the DNA damage response and activated the cGAS-STING-IRF3 signaling pathway to increase CD8+ T cell-recruiting chemokines. Conclusions: Our findings suggested that targeting MYC might eliminate CSCs, prevent metastasis, and activate antitumor immunity to overcome cisplatin resistance in HNSCC.

Interplay between EZH2/β-catenin in stemness of cisplatin-resistant HNSCC and their role as therapeutic targets

The Wnt/β-catenin signaling pathway is associated with the regulation of cancer stem cells, and it can be driven by epigenetic modifications. Here, we aim to identify epigenetic modifications involved in the control of the Wnt/β-catenin signaling and investigate the role of this pathway in the accumulation of cancer stem cells (CSC) and chemoresistance of Head and Neck Squamous Cell Carcinoma (HNSCC). Quantitative-PCR, western blot, shRNA assay, viability assay, flow cytometry assay, spheres formation, xenograft model, and chromatin immunoprecipitation were employed to evaluate the Wnt/β-catenin pathway and EZH2 in wild-type and chemoresistant oral carcinoma cell lines, and in the populations of CSC and non-stem cells. We demonstrated that β-catenin and EZH2 were accumulated in cisplatin-resistant and CSC population. The upstream genes of the Wnt/β-catenin signaling (APC and GSK3β) were decreased, and the downstream gene MMP7 was increased in the chemoresistant cell lines. The inhibition of β-catenin and EZH2 combined effectively decreased the CSC population in vitro and reduced the tumor volume and CSC population in vivo. EZH2 inhibition increased APC and GSK3β, and the Wnt/β-catenin inhibition reduced MMP7 levels. In contrast, EZH2 overexpression decreased APC and GSK3β and increased MMP7. EZH2 and β-catenin inhibitors sensitized chemoresistant cells to cisplatin. EZH2 and H3K27me3 bounded the promoter of APC, leading to its repression. These results suggest that EZH2 regulates β-catenin by inhibiting the upstream gene APC contributing to the accumulation of cancer stem cells and chemoresistance. Moreover, the pharmacological inhibition of the Wnt/β-catenin combined with EZH2 can be an effective strategy for treating HNSCC.

7-Epitaxol induces apoptosis in cisplatin-resistant head and neck squamous cell carcinoma via suppression of AKT and MAPK signalling.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although cisplatin-based chemotherapy is commonly used in HNSCC, frequent development of cisplatin resistance is a potential cause of poor HNSCC prognosis. In the present study, we investigated the anticancer efficacy of a major paclitaxel metabolite namely 7-Epitaxol in cisplatin-resistant HNSCC. The findings revealed that 7-Epitaxol exerts cytotoxic effects in cisplatin-resistant HNSCC cell lines by inducing cell cycle arrest and intrinsic and extrinsic apoptotic pathways. Specifically, 7-Epitaxol increased Fas, TNF-R1, DR5, DcR3 and DcR2 expressions, reduced Bcl-2 and Bcl-XL (anti-apoptotic proteins) expressions, and increased Bid and Bim L/S (pre-apoptotic proteins) expressions, leading to activation of caspase-mediated cancer cell apoptosis. At the upstream cell signalling level, 7-Epitaxol reduced the phosphorylation of AKT, ERK1/2 and p38 to trigger apoptosis. In vivo results showed that animals treated with 7-Epitaxol show antitumor growth compared to control animals. Taken together, the study demonstrates the potential anticancer efficacy of 7-Epitaxol in inducing apoptosis of cisplatin-resistant HNSCC cells through the suppression of AKT and MAPK signalling pathways.

The Vitamin D Receptor-BIM Axis Overcomes Cisplatin Resistance in Head and Neck Cancer.

Simple SummaryTreatment success of head and neck squamous cell carcinoma cancers (HNSCCs) is often hindered by cisplatin resistance. Vitamin D and its receptor (VDR) have been suggested to influence tumor pathobiology and therapy response. We found that VitD/analogs in combination with platinum-based drugs may help to fight therapy resistances in HNSCC. VitD/cisplatin combinations induced E-cadherin upregulation and killed cancer cells by increasing the expression of the pro-apoptotic protein BIM. By identifying the VDR/VitD/BIM axis, we here provide a molecular rationale for the anti-cancer activity of VitD/analogs in combination therapies, which should be further exploited in the clinics.Treatment success of head and neck squamous cell carcinoma (HNSCC) is often hindered by cisplatin resistance. As inherent and acquired therapy resistance counteracts improvement in long-term survival, novel multi-targeting strategies triggering cancer cell apoptosis are urgently required. Here, we identify the vitamin D receptor (VDR) as being significantly overexpressed in tumors of HNSCC patients (n = 604; p = 0.0059), correlating with tumor differentiation (p = 0.0002), HPV status (p = 0.00026), and perineural invasion (p = 0.0087). The VDR, a member of the nuclear receptor superfamily, is activated by its ligand vitamin D (VitD) and analogs, triggering multiple cellular responses. As we found that the VDR was also upregulated in our cisplatin-resistant HNSCC models, we investigated its effect on overcoming cisplatin resistance. We discovered that VitD/cisplatin combinations synergistically killed even cisplatin-resistant cells at clinically achievable levels. Similar results were obtained for the clinically used VitD analog Maxacalcitol. Moreover, VitD/cisplatin combinations inhibited tumor cell migration by E-cadherin upregulation. Signaling pathway analyses revealed that VitD co-treatments triggered cancer cell death by increasing the expression of the pro-apoptotic BCL-2 family protein BIM. BIM’s pro-apoptotic activity in HNSCC cells was confirmed by ectopic overexpression studies. Importantly, BIM expression is positively associated with HNSCC patients’ (n = 539) prognosis, as high expression correlated with improved survival (p = 0.0111), improved therapy response (p = 0.0026), and remission (p = 0.004). Collectively, by identifying, for the first time, the VDR/BIM axis, we here provide a molecular rationale for the reported anti-cancer activity of VitD/analogs in combination therapies. Our data also suggest its exploitation as a potential strategy to overcome cisplatin resistance in HNSCC and other malignancies by inducing additional pro-apoptotic pathways.

Interplay between Partial EMT and Cisplatin Resistance as the Drivers for Recurrence in HNSCC.

This study aims to investigate the role of partial epithelial to mesenchymal transition (pEMT)-related proteins in modulating Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC). SCC-25 cells were pre-treated with TGF-beta1 followed by transient Krüppel-like Factor 4 (KLF4)-overexpression and Cisplatin treatment. Cell growth, cell morphological changes and cell migration were assessed using Juli BR live cell video-microscopy. In addition, Ki-67 and Slug immunostaining and follow-up image cytometric analysis of primary and recurrent HNSCC tumors were performed to evaluate the proliferation index (PI) and the EMT-like phenotype. We observed that proliferating and Slug-positive tumor cells expand after therapy in HNSCC. Subsequently, protein analysis revealed the stabilization of Slug, upregulation of Vimentin and phospho-p38 (p-p38) in Cisplatin-resistant SCC-25 cells. Moreover, KLF4-overexpression contributed to Cisplatin sensitivity by reduction of Slug at the protein level. This work strongly suggests that an pEMT-like pathway is activated in recurrent and Cisplatin-resistant HNSCC. Finally, stable KLF4-overexpression might sensitize HNSCC tumor cells for Cisplatin treatment.

Targeting of c-MET and AXL by cabozantinib is a potential therapeutic strategy for patients with head and neck cell carcinoma

SummaryLocal or metastatic relapse following surgery, radiotherapy, and cisplatin is the leading cause of death in patients with head and neck squamous cell carcinoma (HNSCC). Our study shows overexpression of c-MET and AXL in HNSCC cells and patients resistant to radiotherapy and cisplatin. We demonstrate that cabozantinib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), c-MET, and AXL, decreases migration, invasion, and proliferation and induces mitotic catastrophe and apoptotic cell death of naive and radiotherapy- and cisplatin-resistant HNSCC cells. Cabozantinib inhibits the growth and metastatic spread of experimental HNSCC in zebrafish and the growth of experimental HNSCC in mice by blocking tumor cell proliferation and angiogenesis. The efficacy of cabozantinib is also confirmed on viable sections of surgically removed specimens of human HNSCC and on a patient who relapses after five lines of treatment. These results suggest that cabozantinib is relevant for the treatment of patients with HNSCC after relapse under radiotherapy and cisplatin.

A novel CREB5/TOP1MT axis confers cisplatin resistance through inhibiting mitochondrial apoptosis in head and neck squamous cell carcinoma

BackgroundCisplatin resistance is one of the main causes of treatment failure and death in head and neck squamous cell carcinoma (HNSCC). A more comprehensive understanding of the cisplatin resistance mechanism and the development of effective treatment strategies are urgent.MethodsRNA sequencing, RT-PCR, and immunoblotting were used to identify differentially expressed genes associated with cisplatin resistance. Gain- and loss-of-function experiments were performed to detect the effect of CREB5 on cisplatin resistance and mitochondrial apoptosis in HNSCC. Chromatin immunoprecipitation (ChIP) assay, dual-luciferase reporter assay, and immunoblotting experiments were performed to explore the underlying mechanisms of CREB5.ResultsCREB5 was significantly upregulated in cisplatin-resistant HNSCC (CR-HNSCC) patients, which was correlated with poor prognosis. CREB5 overexpression strikingly facilitated the cisplatin resistance of HNSCC cells in vitro and in vivo, while CREB5 knockdown enhanced cisplatin sensitivity in CR-HNSCC cells. Interestingly, the activation of AKT signaling induced by cisplatin promoted nucleus translocation of CREB5 in CR-HNSCC cells. Furthermore, CREB5 transcriptionally activated TOP1MT expression depending on the canonical motif. Moreover, CREB5 silencing could trigger mitochondrial apoptosis and overcome cisplatin resistance in CR-HNSCC cells, which could be reversed by TOP1MT overexpression. Additionally, double-targeting of CREB5 and TOP1MT could combat cisplatin resistance of HNSCC in vivo.ConclusionsOur findings reveal a novel CREB5/TOP1MT axis conferring cisplatin resistance in HNSCC, which provides a new basis to develop effective strategies for overcoming cisplatin resistance.

Targeting PI3Kα/δ and the ErbB Family of Protein-Tyrosine Kinases in Cisplatin-Resistant Head and Neck Squamous Cell Carcinomas

<h3>Purpose/Objective(s)</h3> The mortality rate for advanced head and neck squamous cell carcinoma (HNSCC) remains very high due to cancer recurrence and metastasis. Cisplatin is a commonly used anti-cancer drug for treatment of recurrent and metastatic HNSCC, but the majority of patients will eventually develop resistance. Therefore, there is an urgent, yet still unmet, need for improved therapies for cisplatin-resistant HNSCC. Increasing evidence has demonstrated that ErbB family kinase and PI3K/Akt are the most important regulators and targets in HNSCC. Our goal is to find new therapeutics to treat cisplatin resistant HNSCC without use of cisplatin. Here, we determined the efficacy of a combination of the PI3Kinase inhibitor, Copanlisib with the ErbB inhibitor, Afatinib, on suppressing cisplatin resistant HNSCC <i>in vitro</i> and <i>in vivo</i>. <h3>Materials/Methods</h3> Both cisplatin sensitive (Cal27 and FaDu) and resistant (Cal27CP and FaDu-CP) HNSCC cells were treated with copanlisib, afatinib, or a combination of copanlisib and afatinib. The effects on cell signaling pathways were tested by Western blot assay and RT-PCR. Cell proliferation was determined by MTT assay. Apoptosis was assayed by Annexin V. Migration and invasion was monitored via the xCELLigence real-time cell system tic cells. Xenograft tumor formation was observed in nude mice. <h3>Results</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals. <h3>Conclusion</h3> We found that the combination of Copanlisib and Afatinib completely blocked PI3K/Akt/mTOR pathways and ErbB kinase family and significantly suppressed cell proliferation, survival, migration, and invasion <i>in vitro</i>, as well as xenograft tumor formation in animals.

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

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. 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. 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. Wee1 inhibitor, AZD1775, and cisplatin coordinately suppressed proliferation and survival of HNSCC.

The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells

Despite major progress in elucidating the pathobiology of head and neck squamous cell carcinoma (HNSCC), the high frequency of disease relapse correlates with unacceptably deficient patient survival. We previously showed that cancer stem-like cells (CSCs) drive tumorigenesis and progression of HNSCC. Although CSCs constitute only 2–5% of total tumor cells, CSCs contribute to tumor progression by virtue of their high tumorigenic potential and their resistance to chemo-, radio-, and immunotherapy. Not only are CSCs resistant to therapy, but cytotoxic agents actually enhance cancer stemness by activating transcription of pluripotency factors and by inducing expression of Bmi-1, a master regulator of stem cell self-renewal. We hypothesized therapeutic inhibition of interleukin-6 receptor (IL-6R) suppresses Bmi-1 to overcome intrinsic chemoresistance of CSCs. We observed that high Bmi-1 expression correlates with decreased (p = 0.04) recurrence-free survival time in HNSCC patients (n = 216). Blockade of IL-6R by lentiviral knockdown or pharmacologic inhibition with a humanized monoclonal antibody (Tocilizumab) is sufficient to inhibit Bmi-1 expression, secondary sphere formation, and to decrease the CSC fraction even in Cisplatin-resistant HNSCC cells. IL-6R inhibition with Tocilizumab abrogates Cisplatin-mediated increase in CSC fraction and induction of Bmi-1 in patient-derived xenograft (PDX) models of HNSCC. Notably, Tocilizumab inhibits Bmi-1 and suppresses growth of xenograft tumors generated with Cisplatin-resistant HNSCC cells. Altogether, these studies demonstrate that therapeutic blockade of IL-6R suppresses Bmi-1 function and inhibits cancer stemness. These results suggest therapeutic inhibition of IL-6R might be a viable strategy to overcome the CSC-mediated chemoresistance typically observed in HNSCC patients.

LncRNA lnc-POP1-1 upregulated by VN1R5 promotes cisplatin resistance in head and neck squamous cell carcinoma through interaction with MCM5

Cisplatin resistance is a major therapeutic challenge in advanced head and neck squamous cell carcinoma (HNSCC). Here, we aimed to investigate the key signaling pathway for cisplatin resistance in HNSCC cells. Vomeronasal type-1 receptor 5 (VN1R5) was identified as a cisplatin resistance-related protein and was highly expressed in cisplatin-resistant HNSCC cells and tissues. The long noncoding RNA (lncRNA) lnc-POP1-1 was confirmed to be a downstream target induced by VN1R5. VN1R5 transcriptionally regulated lnc-POP1-1 expression by activating the specificity protein 1 (Sp1) transcription factor via the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. VN1R5 promoted cisplatin resistance in HNSCC cells in a lnc-POP1-1-dependent manner. Mechanistically, lnc-POP1-1 bound to the minichromosome maintenance deficient 5 (MCM5) protein directly and decelerated MCM5 degradation by inhibiting ubiquitination of the MCM5 protein, which facilitated the repair of DNA damage caused bycisplatin. In summary, we identified the cisplatin resistance-related protein VN1R5 and its downstream target lnc-POP1-1. Upon upregulation by VN1R5, lnc-POP1-1 promotes DNA repair in HNSCC cells through interaction with MCM5 and deceleration of its degradation.

Cabozantinib, a Promising Therapeutic Strategy for Resistant Head and Neck Squamous Cell Carcinoma Patients

Local or metastatic relapses following surgery, radiotherapy and chemotherapy (cisplatin) are the leading causes of death of patients with head and neck squamous cell carcinoma (HNSCC). In the current study, we showed that acquired resistance to radiotherapy and cisplatin of HNSCC cells and patients depends on overexpression of c-MET and AXL. Cabozantinib, a VEGFR, c-MET and AXL inhibitor, decreased migration, invasion and proliferation, and induced mitotic catastrophe and apoptotic cell death of naive and radiotherapy- and cisplatin-resistant HNSCC cells.Cabozantinib inhibited the growth of experimental HNSCC in mice by inhibiting tumor cell proliferation and angiogenesis. Cabozantinib inhibited the growth and the metastatic spread of experimental HNSCC in zebrafish. The efficacy of cabozantinib on experimental models of HNSCC was further confirmed on viable sections of surgically removed specimens of human HNSCC. These results suggest that cabozantinib is highly relevant for the treatment of HNSCC patients following relapses on radiotherapy and cisplatin.

P38 Mitogen-activated protein kinase modulates cisplatin resistance in Head and Neck Squamous Cell Carcinoma cells

ObjectiveThis study aims to investigate the role of p38 Mitogen-activated protein kinase (MAPK) in imparting cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) cells. DesignLaboratory generated cisplatin resistant HNSCC cells were treated with p38 inhibitor and were subjected to increasing dosage of cisplatin. Western blot, immunohistochemistry and RT PCR analysis were performed to investigate expression level of p-p38 and Cancer stem cell (CSC) markers in cisplatin resistant HNSCC cells with or without p38 inhibitor. Chemoresistance, wound healing capacity and Spheroids formation capacity were assessed following p38 inhibition in cisplatin resistant HNSCC cell lines. In addition, alkaline comet assay and γ-H2AX immunostaining were performed to evaluate the DNA damage response and repair abilities in cisplatin resistant HNSCC cells after p38 inhibition. ResultsIt was observed that following p38 inhibition, cisplatin resistant HNSCC cells exhibited significant reduction in expression of CSC markers, β-catenin, reduced migration potential and sphere forming ability along with increased apoptotic index demonstrating there was increased sensitivity towards Cisplatin. Molecular docking study identified several interface amino acid residues between p-p38 with CSC markers (Klf4 and CD44). p38 inhibited cisplatin resistant HNSCC cells also exhibited increased DNA damage as measured by Comet assay and γ-H2AX foci formation index. There was significant decrease in DNA repair as confirmed by reduced ERRC1 expression. ConclusionsOur study demonstrated that p38 MAPK inhibition can be a targeted approach to overcome resistance in HNSCC thereby escalating the effectiveness of chemotherapy in HNSCC.

Inhibition of IKK\u03b2/NF-\u03baB 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 IKKβ/NF-κB in regulation of the sensitivity of cisplatin-resistant HNSCC to Dasatinib. Additionally, we wished to determine whether inhibition of the IKKβ/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 IKKβ/NF-κB signaling in multiple cisplatin-resistant HNSCC. Interestingly, inhibition of IKKβ/NF-κB by CmpdA (Bay65-1942), a recently identified IKKβ 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, IKKβ 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 IKKβ 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 IKKβ play crucial roles in cisplatin-resistant HNSCCC and co-targeting SRC and IKKβ 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 IKKβ inhibition.

We explored the role of the transcription factor, NF-κB, and its upstream kinase IKKβ 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 IKKβ/NF-κB activity compared to their parental partners. Importantly, we found that knockdown of IKKβ, but not NF-κB, dramatically impaired cell migration and invasion in these cells. Consistent with this, the IKKβ 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 IKKβ in control of migration, invasion, and metastasis, and implicated that targeting IKKβ may be a potential therapy for cisplatin-resistant metastatic HNSCC.

Combined treatment with cisplatin and the tankyrase inhibitor XAV-939 increases cytotoxicity, abrogates cancer-stem-like cell phenotype and increases chemosensitivity of head-and-neck squamous-cell carcinoma cells

Cancer stem-like cells (CSCs) were reported to be linked with tumorigenesis, metastasis and resistant to chemo and radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study we investigated the role of CSCs in chemoresistance and abrogation of CSC mediated chemoresistance by combinatorial treatment with cisplatin and small molecule tankyrase inhibitor XAV-939. Two cisplatin-resistant HNSCC cells were generated by stepwise dose incremental strategy. We evaluated the chemoresistance, sphere forming capacity, extent of DNA damage and repair capacity in parental and cisplatin-resistant HNSCC cells. Furthermore, the abrogation of CSC mediated chemoresistance was evaluated in HNSCC cells with XAV-939 alone and in combination with cisplatin. It was observed that cisplatin-resistant HNSCC cell lines exhibited increase in chemoresistance, CSC phenotype and increased DNA repair capacity. We observed that combination of cisplatin and XAV-939 acts synergistically to abrogate chemoresistance by increasing DNA damage. Molecular docking study also revealed similar binding region that could contribute towards synergy predictions between cisplatin and XAV939. In conclusion, this study elucidated that combination of cisplatin and XAV-939 exerted cytotoxic and genotoxic effect to abrogate CSC mediated chemoresistance in HNSCC in synergistic manner.

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

BackgroundWe investigated the role of the ETS-1 transcription factor in Head and Neck Squamous Cell Carcinoma (HNSCC) in multiple cisplatin-resistant HNSCC cell lines.MethodsWe 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.ResultsWe 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.ConclusionsOur results demonstrate that the SRC/ETS-1 pathway plays a crucial role and could be a key therapeutic target in cisplatin-resistant HNSCC treatment.

Inhibition of HDAC6 Protein Enhances Bortezomib-induced Apoptosis in Head and Neck Squamous Cell Carcinoma (HNSCC) by Reducing Autophagy

Chemoresistance is a major barrier to effective chemotherapy of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Recently, autophagy, a highly conservative intracellular recycling system, has shown to be associated with chemoresistance in cancer cells. However, little is known about how autophagy plays a role in the development of chemoresistance in HNSCC and how autophagy is initiated when HNSCC cells undergo cytotoxic stress. Here, we report that autophagy was activated when HNSCC cells are treated with the proteasome inhibitor bortezomib, proposed as an alternative chemotherapeutic agent for both primary and cisplatin-resistant HNSCC cells. Ablation of histone deacetylase 6 (HDAC6) expression and its activity in HNSCC cells significantly inhibited autophagy induction by altering the phosphorylation status of mammalian target of rapamycin and enhanced the bortezomib cytotoxicity. Similarly, a combination regimen of bortezomib and the histone deacetylase inhibitor trichostatin A abolished HDAC6 activity and decreased autophagy induction while significantly enhancing bortezomib-induced apoptosis in HNSCC cells. These data uncover a novel molecular mechanism indicating that HDAC6 may serve as a critical causal link between autophagy, apoptosis, and the cell survival response in HNSCC. A combination regimen resulting in regression of autophagy improves chemotherapeutic efficacy, thereby providing a new strategy to overcome chemoresistance and to improve the treatment and survival of HNSCC patients.