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RHBDD2 is an intramembrane pseudoprotease member of the Rhomboid superfamily. Our previous studies in breast and colorectal cancer indicate an association between RHBDD2 overexpression and advanced tumor stages. Two alternative transcriptional variants have been described for RHBDD2, which would be encoding for different RHBDD2 protein isoforms. The expression of these RHBDD2 variants/isoforms and its association with breast cancer was the focus of this study. First, expression of RHBDD2 splicing variants was evaluated in normal and breast tumor samples. RHBDD2 variant 2 overexpression was detected in tumors in respect to normal breast tissues at the mRNA and protein levels (P<0.05). Moreover, RHBDD2 variant 2 expression was associated with poor prognostic factors such as basal‑like intrinsic subtype (P<0.05), high proliferation (P<0.01) and long‑term risk‑of‑recurrence (P<0.01) scores. Second, the expression of both variants was evaluated under nutritional‑deprived conditions in breast cancer cell lines. Results demonstrated that RHBDD2 splicing was switched from mRNA variant 1 to variant 2 in association with a significant increment of protein isoform B in response to glucose starvation treatment. Therefore, we propose that the switch from the RHBDD2 variant 1, expressed in normal epithelial cells, to variant 2 occurs as an adaptive phenotype to bypass the stressful tumor microenvironment and promote tumor progression. Finally, the RHBDD2 subcellular localization was corroborated at the Golgi apparatus and their associated v‑SNARE transport vesicles, suggesting a putative new role for RHBDD2 in the protein trafficking of human breast cancer cells.

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Rhomboid pseudoproteases are catalytically inactive members of the rhomboid superfamily that modulate the traffic, turnover and activity of their target proteins. Rhomboid domain containing 2 (RHBDD2) is a rhomboid family member overexpressed during mammary gland development and advanced stages of breast cancer. Interactome profiling studies have identified RHBDD2 as a novel binding partner of WW domain-containing oxidoreductase (WWOX) protein.The present study characterized the RHBDD2-WWOX interaction in proliferating and differentiated stages of normal mammary and breast cancer cells by co-immunoprecipitation and confocal microscopy. Normal breast and proliferating cancer cells showed significantly increased RHBDD2 mRNA levels compared with their differentiated counterparts. WWOX mRNA was primarily expressed in differentiated cells. WWOX co-precipitated with RHBDD2, indicating that endogenous RHBDD2 and WWOX were physically associated in normal and breast cancer proliferating cells compared with the differentiated stage. Co-localization assays corroborated the co-immunoprecipitation results, demonstrating the RHBDD2-WWOX protein interaction in normal and proliferating breast cancer cells. RHBDD2 harbors a conserved LPPY motif at the C-terminus region that directly interacted with the WW domains of WWOX. Since WWOX serves as an inhibitor of the TGFβ/SMAD3 signaling pathway in breast cells, modulation of SMAD3 target genes was analyzed in proliferating and differentiated mammary cells and in RHBDD2 silencing assays. Increased expression levels of SMAD3-regulated genes were detected in proliferating cells compared with their differentiated counterparts. Follistatin and angiopoietin-like 4 mRNA was significantly downregulated in RHBDD2 transiently silenced cells compared with scrambled control small interfering RNA. Based on these results, WWOX was suggested to be a novel RHBDD2 target protein involved in the modulation of breast epithelial cell proliferation and differentiation.

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