RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyperrecombination and repair. In this study, we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas, which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence, we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly, siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover, combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably, we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers. Cancer Res; 71(13); 4598-607. Ó2011 AACR.

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) is a tumor suppressor implicated in the regulation of RB1 (retinoblastoma 1) expression. However, the molecular mechanism of RB1 regulation by RB1CC1 has not been elucidated. Here, we demonstrate that nuclear RB1CC1 binds to the RB1 promoter using chromatin immunoprecipitation assays with anti-RB1CC1 antibody. Luciferase assays with RB1 promoter reporter plasmids revealed that RB1CC1 activated the RB1 promoter through the 201 bp upstream GC-rich region (from the initiation ATG). Electrophoretic mobility shift assay and Western blot analysis supported RB1CC1 binding to the GC-rich region of the RB1 promoter. In addition, the C-terminus of RB1CC1 was required for nuclear localization and subsequent RB1 promoter activation. Furthermore, the expression levels of RB1CC1 and RB1 significantly correlated with in vivo breast cancer tissues as determined by immunohistochemical analysis. These data indicate that nuclear RB1CC1 directly activates the RB1 promoter to enhance RB1 expression in cancer cells. Evaluation of RB1CC1 in various types of human cancer tissues is expected to provide useful information for clinical practice and future therapeutic strategies. ' UICCKey words: RB1CC1; RB1; promoter; transcription The RB1-encoded protein (RB1) is a well-known cell cycle regulator, particularly of G1/S-phase cell cycle progression, 1 and also facilitates cellular differentiation. 2 RB1 is believed to be expressed constitutively in various mammalian tissues, though its expression level often varies in a tissue-specific manner. 3 RB1 plays various roles depending on its phosphorylated state, 1 and the regulation of RB1 phosphorylation has been widely studied. However, its transcriptional regulation is poorly understood. 1 Thus, the regulatory mechanism of RB1 expression should be analyzed more thoroughly.RB1-inducible coiled-coil 1 (RB1CC1: the symbol referred to here and approved by the Human Genome Organization [HUGO] Gene Nomenclature Committee; also known as FIP200, focal adhesion kinase family interacting protein of 200 kDa) was identified as an RB1 regulator and was shown to enhance RB1 expression. 3,4 RB1CC1 functions in various processes such as cell growth, 5,6 proliferation, 3,4,7 apoptosis 8 and autophagy. 9 Mutated RB1CC1 lacking wild-type function has been detected in breast cancers, 4,10 and its functional loss resulted in RB1 repression. Moreover, RNAi-mediated knock down of RB1CC1 leads to reduced RB1 levels and resulted in failure of C2C12 myoblast differentiation. 11 However, the molecular mechanism of RB1CC1 in the RB1 pathway has not been precisely determined yet.In our study, the mechanism of RB1CC1 as a transcriptional regulator of RB1 was investigated. Consequently, it was identified that RB1CC1 binds to and activates the RB1 promoter. The C-terminus of RB1CC1 was essential for its nuclear localization and RB1 promoter activation. Additionally, RB1CC1 status significantly correlated with RB1 expression in in vivo human cancers. Material and ...

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG) of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.