Fenretinide, Tocilizumab, and Reparixin Provide Multifaceted Disruption of Oral Squamous Cell Carcinoma Stem Cell Properties: Implications for Tertiary Chemoprevention.

Locoregional recurrence of oral squamous cell carcinoma (OSCC) dramatically reduces patient survival. Further, as many OSCC recurrences are inoperable, radiotherapy and chemotherapy with or without biological adjuncts are the remaining treatment options. Although the tumors may initially respond, radiotherapy- and chemotherapy-resistant cancer stem cells (CSC) can readily repopulate OSCC tumors. Currently, following the initial OSCC treatment, patients are closely monitored until a recurrence or a second primary is detected. Identification of agents with complementary mechanisms to suppress CSC tumorigenic functions could change this passive approach. The goals of this study were twofold: (1) develop and validate CSC-enriched (CSCE) OSCC cell lines and (2) identify chemopreventive agents that obstruct multiple CSCE protumorigenic pathways. CSCE cultures, which were created by paclitaxel treatment followed by three tumorsphere passes, demonstrated CSC characteristics, including increased expression of stem cell and inflammatory genes, increased aldehyde dehydrogenase (ALDH) activity, and enhanced in vitro/in vivo proliferation and invasion. Three chemopreventives, fenretinide, tocilizumab, and reparixin, were selected due to their distinct and complementary CSC-disruptive mechanisms. The CSCE selection process modulated the cells' intermediate filaments resulting in an epithelial-predominant (enhanced cytokeratin, proliferation, IL6 release) line and a mesenchymal-predominant (upregulated vimentin, invasive, IL8 release) line. Our results confirm that 4HPR binds with appreciably higher affinity than Wnt at the Frizzled binding site and significantly inhibits CSC-enabling Wnt-ß-catenin downstream signaling. Notably, combination fenretinide-tocilizumab-reparixin treatment significantly suppressed IL6 and IL8 release, stem cell gene expression, and invasion in these diverse CSCE populations. These promising multiagent in vitro data provide the basis for our upcoming in vivo CSCE tertiary chemoprevention studies.

Benefits of Multifaceted Chemopreventives in the Suppression of the Oral Squamous Cell Carcinoma (OSCC) Tumorigenic Phenotype.

Over one third of patients who have undergone oral squamous cell carcinoma (OSCC) surgical resections develop life-threatening and often untreatable recurrences. A variety of drugs, intended for management of recurrent or disseminated cancers, were designed to exploit cancer cells' reliance upon overexpressed receptors and gratuitous signaling. Despite their conceptual promise, clinical trials showed these agents lacked efficacy and were often toxic. These findings are consistent with evasion of pathway-targeted treatments via extensive signaling redundancies and compensatory mechanisms common to cancers. Optimal secondary OSCC chemoprevention requires long-term efficacy with multifaceted, nontoxic agents. Accordingly, this study evaluated the abilities of three complementary chemopreventives, that is, the vitamin A derivative fenretinide (4-HPR, induces apoptosis and differentiation, inhibits signaling proteins, and invasion), the estrogen metabolite 2-methoxyestradiol (2-ME, apoptosis-inducing, antiangiogenic), and the humanized mAb to the IL6R receptor tocilizumab (TOC, reduces IL6 signaling) to suppress OSCC gratuitous signaling and tumorigenesis. Modeling studies demonstrated 4-HPR's high-affinity binding at STAT3's dimerization site and c-Abl and c-Src ATP-binding kinase sites. Although individual agents suppressed cancer-promoting pathways including STAT3 phosphorylation, STAT3-DNA binding, and production of the trans-signaling enabling sIL6R, maximal chemopreventive effects were observed with agent combinations. OSCC tumor xenograft studies showed that locally delivered TOC, TOC+4-HPR, and TOC+4-HPR+2-ME treatments all prevented significant tumor growth. Notably, the TOC+4-HPR+2-ME treatment resulted in the smallest overall increase in tumor volume. The selected agents use diverse mechanisms to disrupt tumorigenesis at multiple venues, that is, intracellular, tumor cell-ECM, and tumor microenvironment; beneficial qualities for secondary chemopreventives. Cancer Prev Res; 10(1); 76-88. ©2016 AACR.