Conserved oncogenic behavior of the FAM83 family regulates MAPK signaling in human cancer

Abstract

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 elevated tumor grade and decreased overall survival.

Implications: FAM83 proteins represent a novel family of oncogenes suitable for the development of cancer therapies aimed at suppressing MAPK signaling.

Figure 1. The FAM83 proteins have a conserved N-terminal Domain of Unknown Function (DUF1669)

PRALINE amino acid sequence alignment analysis was performed on all 8 FAM83 members (A–H; (11)). The legend indicates the range of conservation from blue (unconserved) to red (conserved), and highlights the highly conserved amino terminus.

Figure 2. Numerous FAM83 members can drive the transformation of HME1 cells

(A) Schematic diagram of the five FAM83 members studied here, illustrating the conserved Domain of Unknown Function (DUF1669), and the number of amino acids encoding each protein. (B–D) HME1 cells were infected with a retrovirus encoding FAM83A-FAM83E, or a control retrovirus (Vector). Immunoblot analysis confirmed the expression of each FAM83 member (B). The FAM83-expressing HME1 cells were plated into soft agar to assess AIG. Images of the colonies were acquired after 3 weeks (C), and the number of colonies was quantified (D). Error bars represent the mean +/− standard deviation for a representative experiment performed in triplicate.

Figure 3. The FAM83 members are overexpressed in diverse human cancers

(A) Origene TissueScan Cancer Panels (384-well multi-cancer survey panel) were analyzed by Real-Time PCR for FAM83A, FAM83B FAM83C, FAM83D, and FAM83E. The sample sizes for each tissue type are as follows: Adrenal: N=2, C=22; Breast: N=2, C=23; Bladder: N=2, C=22; Lung: N=4, C=19; Testis: N=6, C=19; Thyroid: N=3, C=18; Ovary: N=3, C=21; Lymphoid: N=3, C=34. (B) FAM83 members overexpressed in the TissueScan Cancer Panels or in Oncomine microarray data were further examined in additional tissue specific TissueScan Cancer Panels. The sample sizes for each tissue type are as follows: Ovarian: N=13, C=76; Breast: N=16, C=191; Lung: N=38, C=98; Bladder: N=3, C=18. The relative expression of each FAM83 member that had a statistically significant difference between normal, associated (N) and cancerous (C) tissues is presented. The statistical significance was determined using a Welch’s t-test.

Figure 4. Ablation of FAM83A or FAM83D expression suppresses the growth of breast cancer cells

(A) Western analysis (upper panel) or Northern analysis (bottom panel) confirmed elevated expression of FAM83A and FAM83D in MDA468 cancer cells compared to two normal HMEC cell lines (HME). (B) MDA468 cells were infected with lentiviruses encoding an shRNA targeting GFP or two different shRNAs targeting either FAM83A (sh83A2 or sh83A4) or FAM83D (sh83D2 or sh83D5). Western analysis (upper panel) and Real-time analysis (lower panel) confirmed the suppression of FAM83A protein and FAM83D mRNA. (C–E) MDA468 cells expressing control, FAM83A, or FAM83D shRNA were plated to assess relative growth (C), AIG (D), or acini formation in lrBM (E). (F) MDA468 cells expressing control, FAM83A, or FAM83D shRNA were injected subcutaneously into immunocompromised mice to assess tumor formation. Error bars represent the mean +/− standard deviation for a representative experiment performed in triplicate.

Figure 5. Suppression of FAM83A or FAM83D expression inhibits the growth of breast cancer cells and RAS-transformed HMEC

(A–C) HCC1937 cells were infected with lentiviruses encoding an shRNA targeting GFP, FAM83A (sh83A2), FAM83D (sh83D2), or FAM83B (sh83B2). The HCC1937 cells expressing control, FAM83A, or FAM83D shRNA were plated to assess relative growth (A), AIG (B), or acini formation in lrBM (C). (D–E) MDA231 breast cancer cells or HME1 cells transformed by exogenous mutant RAS-G12V (HME1-Ras^G12V) were infected with lentiviruses encoding an shRNA targeting GFP, FAM83A (sh83A2) or FAM83D (sh83D2) and plated to assess relative growth or AIG.

Figure 6. FAM83 members regulate CRAF-MAPK signaling

(A) MDA468 cells were infected with lentiviruses encoding shRNA targeting GFP, FAM83A (sh83A2), FAM83D (sh83D2), or FAM83B (sh83B2), and Western analysis of phospho-ERK1/2, ERK1/2, and GAPDH was performed. (B) FAM83 members co-precipitate CRAF. 293T cells were transfected with an expression constructs encoding CRAF, FAM83A, FAM83C, FAM83D, FAM83E, or a non-coding vector control (V) as indicated. Immunoprecipitation was performed using a FLAG antibody, and precipitated proteins analyzed by Western analysis to determine the amount of CRAF bound to each FAM83 member. (C) Subcellular protein fractionation was performed to isolate cytoplasmic (C) and membrane (M) fractions from HME1 cells expressing GFP, FAM83A, FAM83B, or FAM83D. Normalized portions of each extract were analyzed by Western blotting using antibodies against RAS and CRAF. (D) The relative expression of FAM83A and FAM83D was examined in a panel of 21 matched normal, associated lung (N) and lung cancer (C). Each cancer specimen is normalized to the corresponding normal, which is set equal to 1. (E) Heatmap showing the relative expression of FAM83A, FAM83B, and FAM83D in each tumor from the panel of 21 matched normal, associated lung (N) and lung cancer (C) shown in E. (F) Quantification of the number of tumors overexpressing 0, 1, 2, or all 3 of the FAM83 members by greater than 30-fold is shown for the matched lung dataset. The distribution of each FAM83 member, or pair of FAM83 members is shown within each bar of the graph.