Potential dual functional roles of the Y-linked RBMY in hepatocarcinogenesis

Abstract

Hepatocellular carcinoma (HCC) is a highly heterogeneous liver cancer with significant male biases in incidence, disease progression, and outcomes. Previous studies have suggested that genes on the Y chromosome could be expressed and exert various male-specific functions in the oncogenic processes. In particular, the RNA-binding motif on the Y chromosome (RBMY) gene is frequently activated in HCC and postulated to promote hepatic oncogenesis in patients and animal models. In the present study, immunohistochemical analyses of HCC specimens and data mining of The Cancer Genome Atlas (TCGA) database revealed that high-level RBMY expression is associated with poor prognosis and survival of the patients, suggesting that RBMY could possess oncogenic properties in HCC. To examine the immediate effect(s) of the RBMY overexpression in liver cancer cells, cell proliferation was analyzed on HuH-7 and HepG2 cells. The results unexpectedly showed that RBMY overexpression inhibited cell proliferation in both cell lines as its immediate effect, which led to vast cell death in HuH-7 cells. Transcriptome analysis showed that genes involved in various cell proliferative pathways, such as the RAS/RAF/MAP and PIP3/AKT signaling pathways, were downregulated by RBMY overexpression in HuH-7 cells. Furthermore, in vivo analyses in a mouse liver cancer model using hydrodynamic tail vein injection of constitutively active AKT and RAS oncogenes showed that RBMY abolished HCC development. These findings support the notion that Y-linked RBMY could serve dual tumor-suppressing and tumor-promoting functions, depending on the spatiotemporal and magnitude of its expression during oncogenic processes, thereby contributing to sexual dimorphisms in liver cancer.

Keywords: RBMY; TCGA dataset; Y chromosome; hepatocellular carcinoma; transcriptome analysis.

Figure 1

Expression of RNA‐binding motif, Y (RBMY), testis‐specific protein, Y (TSPY), and the tumor markers glypican 3 (GPC3), Ki‐67, and LIN28B in a representative male hepatocellular carcinoma specimen. The boxed areas in A‐E representing tumor (f‐j) and non‐tumor (k‐o) areas are magnified in (F‐J) and (K‐O) respectively. Nuclei were counterstained by hematoxylin (A‐C, E) and Nuclear Fast Red (D). See text for details. Bar represents 200 μm in (A‐E), 50 μm in (F‐O)

Figure 2

Differential expression of RNA‐binding motif, Y (RBMY) in male hepatocellular carcinoma (HCC) specimens. A, Three types of the RBMY expression patterns: (i) densely positive (left column), (ii) sparsely positive (middle 2 columns), and (iii) negative (right column). Middle row shows magnified images of the boxed areas and bottom row shows negative staining of adjacent nontumor areas. B, Top row, example of RBMY‐densely positive (B1) and Ki‐67 (B2) staining of adjacent sections. B3 and B4 show the magnified images of the boxed area in (B1) and (B2) respectively. C, Example of RBMY‐sparsely positive (C1) and Ki‐67 (C2) staining in the adjacent sections. Bottom panels (C3‐C6) show magnified images of the boxed areas (c3‐c6) on the top panels respectively. Only RBMY‐densely positive tumor cells could be correlated with Ki‐67 expression. Bars represent 100 μm

Figure 3

Expression analysis of RNA‐binding motif, Y (RBMY) and Ki‐67 in the transcriptomes of hepatocellular carcinoma (HCC) specimens in The Cancer Genome Atlas (TCGA) database. A, Plot of RBMY relative expression levels in male HCC transcriptomes, showing the RBMY‐high, RBMY‐low, and RBMY‐negative groups. NT, nontumor tissues; the number in parentheses indicates the respective sample size. B, Kaplan‐Meier survival plot showing the survival rates of the male RBMY‐high (red), male RBMY‐low (orange), and male RBMY‐negative (blue) groups. Log‐rank test P‐values against RBMY‐negative group are indicated. RBMY‐high expression is associated with poor survival of the patients. C, Ratios of RBMY‐high, RBMY‐low, and RBMY‐negative groups at respective HCC pathological stages. Chi‐square test P‐value is indicated. There is a gradual increase (ie from 5% to 20%) in the proportion of RBMY‐high expression level toward later pathologic stages. D, The expression levels of Ki‐67 in the nontumor (NT) and HCC samples of female (left) and male (right) HCC groups negative (−), low (+) and high (++) for RBMY expression respectively. Asterisks indicate t test P‐value < .05. Ki‐67 expression is further elevated in RBMY‐high expression HCC group. E, Kaplan‐Meier survival plot showing the survival rate of the Ki‐67‐high group (orange) and Ki‐67‐low group (green). Log‐rank test P‐value is indicated. High Ki‐67 expression is associated with poor survival of the patients

Figure 4

RNA‐binding motif, Y (RBMY) overexpression inhibits cell proliferation in HuH‐7 (top panels) and HepG2 (bottom panels) cells. A, E, Western blots of EGFP and RBMY in the transduced HuH‐7 (A) and HepG2 cells (E) at 1 d post–Dox induction (+Dox). β‐Actin was used as an internal control. −Dox indicates noninduced cells. B, F, Immunofluorescence showed that the activated RBMY (red, +Dox) was localized in the nuclei of RBMY‐transduced (tet‐ON‐RBMY) HuH‐7 (B) and HepG2 (F) (+Dox). DNA was visualized by DAPI staining (blue). C, G, Cell proliferation assays showed that overexpression of RBMY in HuH‐7 and HepG2 cells significantly inhibited cell proliferation, as compared with EGFP alone (+Dox, right). There was no difference between transduced RBMY and enhanced GFP (EGFP) cells under noninduced conditions (−Dox, left). Asterisks indicate t test P‐value < .05. D, Annexin‐V binding assay at 72 h post–Dox induction showing detached tet‐ON‐RBMY HuH‐7 cells being annexin‐V positive (red), corresponding to dead cells. Bars represent 100 µm in (B, D), and 20 µm in (F)

Figure 5

RNA‐binding motif, Y (RBMY) overexpression abolishes tumor formation in a mouse liver cancer model mediated by constitutively active AKT^myr and NRAS^V12 oncogenes. A, Schematic diagram illustrating the hydrodynamic transfection of the oncogenes in the mouse liver using the Sleeping Beauty (SB) transposon system. DNAs inserted in either pT2 or pT3 vectors are capable of integrating into the hepatocyte genome mediated by SB transposase encoded by the pCMV‐SB plasmid, when they are hydrodynamically co‐injected via the tail vein of the recipient mouse. Using the constitutively active AKT^myr and NRAS^V12 oncogenes, such integration results in transformed hepatocytes that become tumorigenic and develop into loci of hepatocellular carcinoma (HCC) in 8 wk post‐injection. The effects of RBMY in such oncogenic processes are evaluated in this system by inclusion of either pT3‐RBMY or pT3‐EGFP (control) plasmid in the injection mixtures. B, Immunohistochemistry showing the expression (red) of enhanced GFP (EGFP) (anti‐GFP) and RBMY (anti‐RBMY) in the respective transfected livers of the recipients at 3 d post‐injection. C, Gross morphological images of selected livers from AKT^myr/NRAS^V12/EGFP, AKT^myr/NRAS^V12/RBMY, and untreated control mice at 8 wk post‐injection. The constitutively active AKT^myr and NRAS^V12 oncogenes induced foci of tumors with EGFP control plasmid (top row) while inclusion of a RBMY expression vector abolished such tumor formation (middle row) similar to untreated controls (bottom row). Bar represents 1 cm. D, Average liver weight of the mice corresponding to the results of an experiment, as presented in (C). Asterisk indicates Mann‐Whitney test P‐value < .05, and numbers in parentheses indicate the respective sample size. Bar indicates the standard error of each group. ND, no difference