Aberrant regulation of growth signaling is a hallmark of cancer development that often occurs through the constitutive activation of growth factor receptors or their downstream effectors. Using validation-based insertional mutagenesis (VBIM), we identified family with sequence similarity 83, member B (FAM83B), based on its ability to substitute for RAS in the transformation of immortalized human mammary epithelial cells (HMECs). We found that FAM83B coprecipitated with a downstream effector of RAS, CRAF. Binding of FAM83B with CRAF disrupted CRAF/14-3-3 interactions and increased CRAF membrane localization, resulting in elevated MAPK and mammalian target of rapamycin (mTOR) signaling. Ablation of FAM83B inhibited the proliferation and malignant phenotype of tumor-derived cells or RAS-transformed HMECs, implicating FAM83B as a key intermediary in EGFR/RAS/MAPK signaling. Analysis of human tumor specimens revealed that FAM83B expression was significantly elevated in cancer and was associated with specific cancer subtypes, increased tumor grade, and decreased overall survival. Cumulatively, these results suggest that FAM83B is an oncogene and potentially represents a new target for therapeutic intervention. IntroductionThe discovery of targets suitable for the development of specific and effective anticancer therapies remains one of the principal challenges facing cancer research. The identification of genes involved in tumorigenesis is essential for devising new targeted therapeutics and can be greatly facilitated by phenotypic-based forward genetic screens for mutations contributing to malignant transformation in human cell models. We recently created a validation-based insertional mutagenesis (VBIM) strategy that expands the application of reversible promoter insertion to nearly any type of mammalian cell (1). The VBIM strategy uses the unique transcriptomes of different human epithelial cell types and provides opportunities for the identification of tissue-specific oncogenes and tumor suppressors. The VBIM lentiviruses alter the unique transcriptome of the model system by introducing promoters into the genome, resulting in dominant genetic alterations that increase the expression of sequences neighboring the insertion sites. By using Cre recombinase-mediated excision of the VBIM promoter, one can revert the VBIM-specific mutants and distinguish them from spontaneous mutants, allowing spontaneous mutants to be eliminated from further study.We have used the VBIM strategy to identify family with sequence similarity 83, member B (FAM83B), as a putative oncogene capable of promoting the transformation of immortalized human mammary epithelial cells (HMECs). We demonstrated that elevated FAM83B expression stimulated aberrant activation of MAPK signaling by altering binding of regulatory 14-3-3 proteins to CRAF and increasing CRAF membrane localization. In addition to driving cellular transformation, FAM83B mRNA was significantly elevated in many human tumor tissues. Ablation of FAM83B from breast cancer cells with ele...

Resistance to chemotherapy remains a major barrier to the successful treatment of cancer. To understand mechanisms underlying docetaxel resistance in breast cancer, we used an insertional mutagenesis strategy to identify proteins whose overexpression confers resistance. A strong promoter was inserted approximately randomly into the genomes of tumorderived breast cancer cells, using a novel lentiviral vector. We isolated a docetaxel-resistant clone in which the level of the kinesin KIFC3 was elevated. When KIFC3 or the additional kinesins KIFC1, KIF1A, or KIF5A were overexpressed in the breast cancer cell lines MDA-MB231 and MDA-MB 468, the cells became more resistant to docetaxel. The binding of kinesins to microtubules opposes the stabilizing effect of docetaxel that prevents cytokinesis and leads to apoptosis. Our finding that kinesins can mediate docetaxel resistance might lead to novel therapeutic approaches in which kinesin inhibitors are paired with taxanes. [Cancer Res 2009;69(20):8035-42]

FAM83B (Family with sequence similarity 83, member B) was recently identified as a novel oncogene involved in activating CRAF/MAPK signaling and driving epithelial cell transformation. FAM83B is one of eight members of a protein family (FAM83) characterized by a highly conserved domain of unknown function (DUF1669), which is necessary and sufficient to drive transformation. Here, it is demonstrated that additional FAM83 members also exhibit oncogenic properties and have significantly elevated levels of expression in multiple human tumor types using a TissueScan Cancer Survey Panel PCR array and database mining. Furthermore, modeling the observed tumor expression of FAM83A, FAM83C, FAM83D, or FAM83E promoted human mammary epithelial cell (HMEC) transformation, which correlated with the ability of each FAM83 member to bind CRAF (RAF1) and promote CRAF membrane localization. Conversely, ablation of FAM83A or FAM83D from breast cancer cells resulted in diminished MAPK signaling with marked suppression of growth in vitro and tumorigenicity in vivo. Importantly, each FAM83 member was determined to be elevated in at least one of 17 distinct tumor types examined, with FAM83A, FAM83B, and FAM83D most frequently overexpressed in several diverse tissue types. Finally, evidence suggests that elevated expression of FAM83 members is associated with tumor grade and overall survival. Implications FAM83 proteins represent a novel family of oncogenes suitable for the development of cancer therapies aimed at suppressing MAPK signaling.

Oncogene-induced senescence (OIS), the proliferative arrest engaged in response to persistent oncogene activation, serves as an important tumor-suppressive barrier. We show here that finite lifespan human mammary epithelial cells (HMEC) undergo a p16/ RB-and p53-independent OIS in response to oncogenic RAS that requires TGF-β signaling. Suppression of TGF-β signaling by expression of a dominant-negative TGF-β type II receptor, use of a TGF-β type I receptor inhibitor, or ectopic expression of MYC permitted continued proliferation upon RAS expression. Surprisingly, unlike fibroblasts, shRNA-mediated knockdown of ATM or CHK2 was unable to prevent RAS-mediated OIS, arguing that the DNA damage response is not required for OIS in HMEC. Abrogation of TGF-β signaling not only allowed HMEC lacking p53 to tolerate oncogenic RAS but also conferred the capacity for anchorage-independent growth. Thus, the OIS engaged after dysregulated RAS expression provides an early barrier to malignant progression and is mediated by TGF-β receptor activation in HMEC. Understanding the mechanisms that initiate and maintain OIS in epithelial cells may provide a foundation for future therapies aimed at reengaging this proliferative barrier as a cancer therapy. (ii) constitutive growth signaling, (iii) unlimited replication potential, and (iv) invasive potential (1). Early studies using normal mouse cells indicated that a limited set of genetic manipulations could confer neoplastic potential (2). However, normal human cells have been more difficult to transform to malignancy, indicative of their more stringent tumor-suppressive pathways. Extensive study of cultured human mammary epithelial cells (HMEC) has identified two senescence barriers. One involves the stress-associated induction of the cyclin-dependent kinase inhibitor p16 before attaining critically short telomeres. This stasis barrier can be overcome by inhibiting p16, allowing continued proliferation, which results in agonescence, a proliferative barrier mediated by telomere depletion (3). Additionally, the ability of dysregulated oncogenic signaling to induce senescence in human cells has implicated oncogene-induced senescence (OIS) as an important tumor-suppressive barrier. A number of recent studies have demonstrated the physiological relevance of OIS in human tumorigenesis and in vivo tumor mouse models (4). Additionally, the presence of senescent cells in benign but not advanced tumors argues that OIS serves as an early tumorsuppressive barrier that needs to be dismantled for full oncogenic progression (4). In human fibroblasts, OIS could be bypassed by disabling p16 or molecular components of the DNA damage response (DDR), including ATM, CHK2, or p53, before RAS, MOS, or STAT5 overexpression (5-9). However, OIS in HMEC has been shown to be independent of p53 and the p16-RB pathway after oncogenic RAF-1 expression (10). The contrasting responses between epithelial and fibroblast cells argue that the signaling networks responsible for OIS have tissue specificity. Indeed, fibrob...

Cytokines play an important role in creating an inflammatory microenvironment, which is now considered a hallmark of cancer. While tumor cells can exploit cytokine signaling to promote growth, invasion and metastasis, the response of normal and premalignant epithelial cells to cytokines present in a developing tumor microenvironment remains unclear. Oncostatin M (OSM), an IL-6 family cytokine responsible for STAT3 activation, has been implicated in cancer development, progression, invasion and metastasis. Paradoxically, OSM can also suppress the growth of normal cells and certain tumor-derived cell lines. Using isogenic human mammary epithelial cells (HMEC) at different stages of neoplastic transformation, we found that OSM signaling suppressed c-MYC expression and engaged a p16- and p53-independent growth arrest that required STAT3 activity. Inhibition of STAT3 activation by expressing a dominant-negative STAT3 protein or a STAT3-shRNA prevented the OSM-mediated arrest. In addition, expression of c-MYC from a constitutive promoter also abrogated the STAT3-mediated arrest, and strikingly, cooperated with OSM to promote anchorage-independent growth (AIG), a property associated with malignant transformation. Cooperative transformation by c-MYC and OSM required PI3K and AKT signaling, demonstrating the importance of multiple signaling pathways downstream of the OSM receptor in defining the cellular response to cytokines. These findings identify c-MYC as an important molecular switch that alters the cellular response to OSM-mediated signaling from tumor suppressive to tumor promoting.