Cisplatin-resistant Head And Neck Squamous Cell Carcinoma Research Articles

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.

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 IKKβ 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.

Abstract 5474: PD-L1 regulates cisplatin chemoresistance in head and neck squamous cell carcinoma

Abstract PD-L1 has been known to play a major role in suppressing the immune system. The effect of PD-L1 has been clinically studied by inhibiting PD-L1 in tumor and tumor-infiltrating immune cells, thereby activating anti-tumor T cells and re-establishing anti-tumor immune responses. Little is known about a unique role of PD-L1 in cisplatin chemoresistance of head and neck squamous cell carcinoma (HNSCC). The aim of the current study is to investigate a novel finding that PD-L1 regulates DNA double strand break (DSB) repairs via the MRE11-RAD50-NBS1 (MRN) complex and contributes to cisplatin chemoresistance in HNSCC. Well-characterized HNSCC tumor cell lines were used for this study. JHU 006 tumor cell line has a cisplatin resistant phenotype whereas JHU 020 is cisplatin sensitive. PD-L1 expression was determined by quantitative polymerase chain reaction (qPCR) and western blot before and after cisplatin treatment. The interactions between PD-L1 and MRN in DNA DSB mediated chemoresistant pathway were studied. PD-L1 siRNA was used to induce chemosensitization of HNSCC cells to cisplatin. Our study demonstrates that treatment of HNSCC cells with cisplatin resulted in increased expression of PD-L1 in cisplatin resistant tumor cells, whereas chemosensitive tumor cells showed no change in PD-L1 expression. Additionally, knockdown of PD-L1 using siRNA resulted in downregulation of NBS1, a key protein of the MRN DNA DSB repair complex. However, NBS1 was able to modulate the levels of PD-L1 expression as the use of either NBS1 siRNA or cells that had been transfected with a dominant negative NBS1 mutant resulted in increased PD-L1 expression. The experimental data suggest that PD-L1 may act as an upstream regulator of NBS1 and may facilitate formation of the MRN complex, enhancing repair of cisplatin induced DNA damage, thereby increasing the cell's resistance to chemotherapeutics. Decreasing expression of PD-L1 via siRNA leads to increased sensitivity of HNSCC cells to cisplatin. This highlights the importance of PD-L1 in the development of chemoresitance and suggests a potential therapeutic strategy for patients with cisplatin-resistant HNSCC. In conclusion, we have identified a novel role for PD-L1 in the development of chemoresistance through its interaction with the MRN complex, which plays a major role in the repair of chemo-induced DNA DSB. Therapies aimed at blocking PD-L1 show great promise because of their ability to enhance both chemotherapeutics and the anti-tumor immune response. Citation Format: Randall S. Ruffner, Andrew Ramsey, Bert W. O'Malley, Daqing Li. PD-L1 regulates cisplatin chemoresistance in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2015-5474

Liposome encapsulated curcumin-difluorinated (CDF) inhibits the growth of cisplatin resistant head and neck cancer stem cells.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with 600,000 new cases every year worldwide. Although chemotherapeutics exist, five-year survival is only 50%. New strategies to overcome drug resistance are required to improve HNSCC treatment. Curcumin-difluorinated (CDF), a synthetic analog of curcumin, was packaged in liposomes and used to evaluate growth inhibition of cisplatin resistant HNSCC cell lines CCL-23R and UM-SCC-1R generated from the parental cell lines CCL-23 and UM-SCC-1 respectively. Growth inhibition in vitro and expression levels of the CD44 (cancer stem cell marker), cytokines, and growth factors were investigated after liposomal CDF treatment. The in vivo growth inhibitory effect of liposomal CDF was evaluated in the nude mice xenograft tumor model of UM-SCC-1R and the inhibition of CD44 was measured. Treatment of the resistant cell lines in vitro with liposomal CDF resulted in a statistically significant growth inhibition (p < 0.05). The nude mice xenograft study showed a statistically significant tumor growth inhibition of UM-SCC-1R cells and a reduction in the expression of CD44 (p < 0.05), indicating an inhibitory effect of liposomal CDF on CSCs. Our results demonstrate that delivery of CDF through liposomes may be an effective method for the treatment of cisplatin resistant HNSCC.

TTP mediates cisplatin-induced apoptosis of head and neck cancer cells by down-regulating the expression of Bcl-2

The chemotherapeutic agent cisplatin is widely used for treatment of head and neck squamous cell carcinoma (HNSCC). B-cell lymphoma 2 (Bcl-2) is an anti-apoptotic protein that is overexpressed in cancer cells and confers resistance to cisplatin. Thus, inhibition of Bcl-2 expression may enhance the cisplatin sensitivity of cancer cells. In this study, we report that the AU-rich element (ARE) binding protein tristetraprolin (TTP) inhibits the expression of Bcl-2 and enhances cisplatin sensitivity of HNSCC cells. Cisplatin-sensitive HNSCC cells express high levels of TTP and low levels of Bcl-2, while cisplatin-resistant HNSCC cells have low levels of TTP and high levels of Bcl-2. Inhibition of TTP expression using siRNA increases levels of Bcl-2 and decreases cisplatin sensitivity in HNSCC cells. On the contrary, overexpression of TTP decreases Bcl-2 expression and increases sensitivity to cisplatin. Together, the results of the present study suggest that TTP expression enhances cisplatin sensitivity in HNSCC cells by reducing levels of Bcl-2.

Decitabine rescues cisplatin resistance in head and neck squamous cell carcinoma.

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) reduces survival. In this study we hypothesized that methylation of key genes mediates cisplatin resistance. We determined whether a demethylating drug, decitabine, could augment the anti-proliferative and apoptotic effects of cisplatin on SCC-25/CP, a cisplatin-resistant tongue SCC cell line. We showed that decitabine treatment restored cisplatin sensitivity in SCC-25/CP and significantly reduced the cisplatin dose required to induce apoptosis. We then created a xenograft model with SCC-25/CP and determined that decitabine and cisplatin combination treatment resulted in significantly reduced tumor growth and mechanical allodynia compared to control. To establish a gene classifier we quantified methylation in cancer tissue of cisplatin-sensitive and cisplatin-resistant HNSCC patients. Cisplatin-sensitive and cisplatin-resistant patient tumors had distinct methylation profiles. When we quantified methylation and expression of genes in the classifier in HNSCC cells in vitro, we showed that decitabine treatment of cisplatin-resistant HNSCC cells reversed methylation and gene expression toward a cisplatin-sensitive profile. The study provides direct evidence that decitabine restores cisplatin sensitivity in in vitro and in vivo models of HNSCC. Combination treatment of cisplatin and decitabine significantly reduces HNSCC growth and HNSCC pain. Furthermore, gene methylation could be used as a biomarker of cisplatin-resistance.

SERPINB2 down‐regulation contributes to chemoresistance in head and neck cancer

Resistance to cisplatin-based chemotherapy is responsible for the majority of deaths from head and neck squamous cell carcinoma (HNSCC). In this study, using genome-wide gene expression analysis to investigate potential molecular mediators of HNSCC chemoresistance, we identified SERPINB2, a known inhibitor of extracellular serine proteinase urokinase-type plasminogen activator (uPA), as an important candidate. Whereas SERPINB2 is known to function as a suppressor of uPA molecular cascades, many of which play important roles in tumor invasion and metastasis, a role for SERPINB2 in cancer drug resistance has not been examined. By using quantitative real-time PCR and Western blot analysis, we determined that SERPINB2 mRNA and protein levels correlated with chemoresistance in HNSCC cell lines, and significantly lower SERPINB2 expression levels were observed in two cisplatin resistant HNSCC subclones compared to their isogenic drug-sensitive parental lines. Immunohistochemical analysis of HNSCC tumor tissues from patients treated with neoadjuvant cisplatin-based chemotherapy (n = 67 cases) revealed a significant association between SERPINB2 protein levels, tumor differentiation and patient relapse. Moreover, SERPINB2 down-regulation was a strong predictor of reduced overall survival in patients with HNSCC who received cisplatin-based chemotherapy (P = 0.001, log rank test). Studies using either siRNA-mediated down-regulation or forced over-expression of SERPINB2 in HNSCC cell lines confirmed a functional role for SERPINB2 in drug resistance. The findings were further supported using chemical inhibitors of STAT3 activity (a downstream effecter of uPAR signaling pathway), showing that STAT3 suppression altered HNSCC cell line cisplatin sensitivity. This is the first report on a role for SERPINB2 in acquired resistance to cisplatin in patients with HNSCC.

Overexpression of CD147 contributes to the chemoresistance of head and neck squamous cell carcinoma cells

Head and neck cancer is the sixth most common cancer in the world and most of them are squamous cell carcinomas. High frequency of cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) leads to tumor relapse and irresponsiveness to cisplatin-based chemotherapy. However, the mechanisms underlying cisplatin resistance is still largely unrevealed. In this study, we found that CD147 was overexpressed in cisplatin-resistant HNSCC cell lines. Based on the result, CD147 expression was down-regulated in the cisplatin-resistant cell line and we observed that the sensitivity to cisplatin increased, as showed in the results of MTT assay and PI-staining apoptotic detection. Meanwhile, transfection of CD147 expression vector promoted the occurrence of cisplatin resistance in the cisplatin-sensitive cell line. Simultaneously blocking of uPAR with neutralizing antibody would significantly prevent the occurrence of cisplatin resistance induced by CD147 overexpression. In conclusion, our study finds that CD147 is also involved in mediating cisplatin resistance in HNSCC and uPAR serves as a possible candidate that collaborates with CD147 in this process.

Targeting Apoptosis to Overcome Cisplatin Resistance: A Translational Study in Head and Neck Cancer

Cisplatin resistance remains a barrier to organ-sparing and survival of patients with advanced head and neck squamous cell carcinoma (HNSCC). Targeted therapies to overcome cisplatin-resistant HNSCC are being developed. Cisplatin-sensitive parental HNSCC cell lines and cisplatin-resistant progeny were studied. Pretreatment HNSCC biopsies were used to construct tissue microarrays which were stained for p53 and Bcl-xL. HNSCC cell lines selected for cisplatin resistance had wild-type p53 and high levels of Bcl-xL. Expression of wild-type p53 in cell lines with low Bcl-xL enhanced cisplatin sensitivity. Expression of both Bcl-xL and wild-type p53 caused tumor cells to become cisplatin resistant. Patients whose tumors expressed low levels of p53 and Bcl-xL enjoyed the best organ preservation and disease-free survival whereas patients whose tumors expressed low levels of p53 and high levels of Bcl-xL had the worst outcome. Novel agents that inhibit Bcl-xL or activate p53 function may target cisplatin-resistant HNSCC. Cisplatin resistance in HNSCC is mediated, at least in part, by high Bcl-xL and functional p53.

Docetaxel Associated Pathways in Cisplatin Resistant Head and Neck Squamous Cell Carcinoma: A Pilot Study

This is a pilot study to identify changes in gene and protein expressions after treatment with docetaxel in cisplatin-resistant head and neck squamous cell carcinoma (HNSCC). Two cisplatin-resistant HNSCC cell lines, HN30 and HN12, were treated with either docetaxel or cisplatin. After 48 hours, differential gene expression between the two treatment groups (docetaxel-treated cells and cisplatin-treated cells) was analyzed using cDNA microarray. Differential protein expression between these two treatment groups was determined using PowerBlot and Western Blot analysis A total of 150 genes and proteins were found to have differential expression patterns in HNSCC after treatment with docetaxel versus cisplatin. Many of these differentially expressed genes and proteins were involved in the cell cycle (decreased E2F), apoptosis (increased bax), angiogenesis (increased thrombospondin), and signal transduction (decreased epidermoid growth factor receptor) regulatory pathways. Gene and protein expression are different and distinct between cells treated with docetaxel and cells treated with cisplatin. This finding provides evidence that different molecular pathways leading to cell death are targeted by docetaxel and cisplatin. Future studies focusing on these differentially expressed genes and proteins may improve our understanding, at the molecular level, of the mechanisms responsible for docetaxel-induced apoptosis in cisplatin-resistant HNSCC. Furthermore, these differentially expressed genes and proteins can be exploited as useful surrogate endpoint biomarkers in future clinical trials using docetaxel.