Background Despite the introduction of several novel therapeutic approaches that improved survival, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease. Herein we report the synergistic antitumor interaction between two well-known drugs used for years in clinical practice, the antiepileptic agent with histone deacetylase inhibitory activity valproic acid and the cholesterol lowering agent simvastatin, in mCRPC models. Methods Synergistic anti-tumor effect was assessed on PC3, 22Rv1, DU145, DU145R80, LNCaP prostate cancer cell lines and EPN normal prostate epithelial cells, by calculating combination index (CI), caspase 3/7 activation and colony formation assays as well as on tumor spheroids and microtissues scored with luminescence 3D-cell viability assay. Cancer stem cells (CSC) compartment was studied evaluating specific markers by RT-PCR, western blotting and flow cytometry as well as by limiting dilution assay. Cholesterol content was evaluated by 1H-NMR. Overexpression of wild-type YAP and constitutively active YAP5SA were obtained by lipofectamine-based transfection and evaluated by immunofluorescence, western blotting and RT-PCR. 22Rv1 R_39 docetaxel resistant cells were selected by stepwise exposure to increasing drug concentrations. In vivo experiments were performed on xenograft models of DU145R80, 22Rv1 parental and docetaxel resistant cells, in athymic mice. Results We demonstrated the capacity of the combined approach to target CSC compartment by a novel molecular mechanism based on the inhibition of YAP oncogene via concurrent modulation of mevalonate pathway and AMPK. Because both CSCs and YAP activation have been associated with chemo-resistance, we tested if the combined approach can potentiate docetaxel, a standard of care in mCRCP treatment. Indeed, we demonstrated, both in vitro and in vivo models, the ability of valproic acid/simvastatin combination to sensitize mCRPC cells to docetaxel and to revert docetaxel-resistance, by mevalonate pathway/YAP axis modulation. Conclusion Overall, mCRPC progression and therapeutic resistance driven by CSCs via YAP, can be tackled by the combined repurposing of two generic and safe drugs, an approach that warrants further clinical development in this disease.
Recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) is a devastating malignancy with a poor prognosis. The combination of cisplatin (CDDP) plus cetuximab (CX) is one of the standard first-line treatments in this disease. However, this therapeutic regimen is often associated with high toxicity and resistance, suggesting that new combinatorial strategies are needed to improve its therapeutic index. In our study, we evaluated the antitumor effects of valproic acid (VPA), a well-known antiepileptic agent with histone deacetylase inhibitory activity, in combination with CDDP/CX doublet in head and neck squamous cell carcinoma (HNSCC) models. We demonstrated, in HNSCC cell lines, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX resulted in a clear synergistic antiproliferative and pro-apoptotic effects. The synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, suggesting the ability of the combined approach to affect also the cancer stem cells compartment. Mechanistically, VPA enhanced DNA damage in combination treatment by reducing the mRNA expression of ERCC Excision Repair 1, a critical player in DNA repair, and by increasing CDDP intracellular concentration via upregulation at transcriptional level of CDDP influx channel copper transporter 1 and downregulation of the ATPAse ATP7B involved in CDDP-export. Valproic acid also induced a dose-dependent downregulation of epidermal growth factor receptor (EGFR) expression and of MAPK and AKT downstream signaling pathways and prevent CDDP- and/or CX-induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of genes induced by non-canonical activity of nuclear EGFR, such as cyclin D1 and thymidylate synthase. Finally, we confirmed the synergistic antitumor effect also in vivo in both heterotopic and orthotopic models, demonstrating that the combined treatment completely blocked HNSCC xenograft tumors growth in nude mice. Overall, the introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. A phase II clinical trial exploring the combination of VPA and CDDP/CX in R/M HNSCC patients is currently ongoing in our institute.
Although fluoropyrimidines were introduced as anticancer agents over 60 years ago, they are still the backbone of many combination chemotherapy regimens for the treatment of solid cancers. Like other chemotherapeutic agents, the therapeutic efficacy of fluoropyrimidines can be affected by drug resistance and severe toxicities; thus, novel therapeutic approaches are required to potentiate their efficacy and overcome drug resistance. In the last 20 years, the deregulation of epigenetic mechanisms has been shown to contribute to cancer hallmarks. Histone modifications play an important role in directing the transcriptional machinery and therefore represent interesting druggable targets. In this review, we focused on histone deacetylase inhibitors (HDACis) that can increase antitumor efficacy and overcome resistance to fluoropyrimidines by targeting specific genes or proteins. Our preclinical data showed a strong synergistic interaction between HDACi and fluoropyrimidines in different cancer models, but the clinical studies did not seem to confirm these observations. Most likely, the introduction of increasingly complex preclinical models, both in vitro and in vivo, cannot recapitulate human complexity; however, our analysis of clinical studies revealed that most of them were designed without a mechanistic approach and, importantly, without careful patient selection.
INTRODUCTION. Development of immune-check-point inhibitors (ICI) has greatly changed the treatment of recurrent or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) demonstrating durable antitumor response and overall survival (OS) improvement. However, more than 70% of patients do not respond to ICI and, recently, phase III Checkmate 651 trial did not demonstrate OS improvement of first-line ICI vs standard EXTREME regimen (cetuximab (CX) plus 5-fluorouracil/cisplatin (CDDP) in R/M HNSCC patients (ESMO2021-LBA36). We have previously showed in vitro and in vivo models of HNSCC the synergistic antitumor interaction of CDDP/CX combined with the antiepileptic valproic acid (VPA), a histone deacetylase inhibitor (HDACi) with anticancer and immunomodulatory properties (Iannelli F., 2020). Here we investigate the immunomodulatory effects of VPA/CDDP/CX combination, focusing on the induction of "immunogenic cell death" (ICD)-like features. METHODS. ICD-related damage-associated molecular patterns (DAMPs) calreticulin (CARL), ATP, HMGB1 and Annexin-A1 (ANXA1) expression was evaluated by western blot, immunofluorescence and/or bioluminescent assays on HNSCC Cal27 and osteosarcoma U2OS cells. Maturation, migration, cytokines release, and phagocytic capacity of dendritic cells (IL4_DCs) was evaluated incubating IL4-DCs with conditioned medium from untreated/treated Cal27 cells and/or in co-culture IL4-DCs/Cal27 assays by ELISA, cytofluorimetric assays and confocal microscopy. RESULTS. VPA/CDDP/CX combination induced in both U2OS and Cal27 cells, synergistic pre-apoptotic exposure of CARL along with HMGB1, ANXA1 and ATP release as compared with single drugs or doublet combination. Moreover, the triple combination increases IL4-DCs migration towards pretreated Cal27 cells as well as the rate of IL4-DCs phagocytosis of apoptotic bodies released by Cal27 dying cells. Triple combination also induces phenotypical maturation of DCs as shown by the increased surface expression of CD83 and CD86. Furthermore, conditioned medium from VPA/CDDP/CX treated Cal27 cells, induced increased release of immune-stimulating cytokines and chemokines IL2, IL1β, TNFα, MIP-1α and G-CSF by IL4-DCs as well as the impairment of immunosuppressive cytokines release IL-10 and VEGF, by VPA/CDDP/CX treated Cal27, co-cultured with IL4-DCs. In vivo experiments in immunocompetent syngeneic mouse model using HNSCC AT-84 cells transduced with human-EGFR are ongoing. CONCLUSION. Overall, we demonstrated that VPA/CDDP/CX is able to enhance the immunogenicity of HNSCC model by inducing ICD. This mechanistic hypothesis will be tested by assessing ICD biomarkers on samples of patients enrolled in the V-CHANCE phase 2 clinical trial evaluating the CDDP/CX associated with VPA in R/M SCCHN patients and recently concluded (NCT02624128). Citation Format: Federica Iannelli, Andrea Ilaria Zotti, Maria Serena Roca, Laura Grumetti, tania Moccia, carlo vitagliano, susan costantini, francesca capone, francesca collina, Lucia Gabriele, stefania parlato, Giulia Romagnoli, Oliver Kepp, Guido Kroemer, Elena Di Gennaro, Alfredo Budillon. Immunomodulatory effects of valproic acid in combination with cisplatin and cetuximab in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1353.
INTRODUCTION: Recurrent metastatic (R/M) squamous cell carcinoma of the head and neck (HNSCC) is a devastating malignancy with a poor prognosis and the combination of cisplatin (CDDP) plus cetuximab (CX) is one of the gold standard for first-line treatment. However, this therapy is often associated with toxicity and resistance, suggesting that new combinatorial strategies are needed to improve the therapeutic index of this regimen. In our study, we evaluated the synergistic antitumor effect of valproic acid (VPA), an anticonvulsant compound with histone deacetylase inhibitor (HDACi) activity, in combination with CDDP/CX in HNSCC models in vitro and in vivo. METHODS: Synergistic anti-proliferative effects were assessed on HNSCC Cal27, FaDu and Cal33 cell lines and BJ-hTERT normal fibroblast, by calculating combination index (CI) accordingly to Chou and Talalay method. Apoptosis was measured by flow cytometry analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. In vivo experiment was performed on xenograft models in athymic mice. RESULTS: We demonstrated, in HNSCC cells, but not in normal human fibroblasts, that simultaneous exposure to equitoxic doses of VPA plus CDDP/CX results in a clear synergistic antiproliferative and proapoptotic effect. Interestingly, in order to better recapitulate tumor growth complexity compared to 2D monolayers conditions, the synergistic antitumor effect was confirmed in four different 3D-self-assembled spheroid models, as well as in in vivo Cal27 xenograft model. Mechanistically, VPA induced a dose-dependent down-regulation of EGFR expression/activation affecting its downstream canonical pathway (pAKT and pMAPK), which plays a driver role in HNSCC. Moreover, we demonstrated that VPA was able to prevent the CDDP and/or CX induced EGFR nuclear translocation, a well-known mechanism of resistance to chemotherapy. Indeed, VPA impaired the transcription of cyclin D1 and DNA repair genes, regulated by non-canonical activity of nuclear EGFR, thus increasing DNA damage induced by CDDP/CX combination. Moreover, VPA was able to enhance the sensitivity to CDDP, by upregulating, at transcriptional level, the CDDP influx channel copper transporter 1 (CTR1). CONCLUSIONS: The introduction of a safe and generic drug such as VPA into the conventional treatment for R/M HNSCC, represents an innovative and feasible antitumor strategy that warrants further clinical evaluation. Indeed, we are currently enrolling patients in a phase-2 clinical trial in order to explore whether the addition of VPA to the standard combination CDDP/CX can increase the response rate in patients with R/M HNSCC. Citation Format: Federica Iannelli, Andrea Ilaria Zotti, Maria Serena Roca, Laura Grumetti, Tania Moccia, Carlo Vitagliano, Chiara Ciardiello, Francesca Bruzzese, Alessandra Leone, Francesco Caponigro, Franco Ionna, Francesco Longo, Elena Di Gennaro, Alfredo Budillon. Valproic acid, by preventing cisplatin/cetuximab-induced EGFR nuclear translocation and increasing cisplatin uptake, potentiates the antitumor effect of the combination treatment in head and neck squamous cell carcinomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5223.
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