SH3GL1 expression is increased in liver cancer and liver CSCs
Firstly, public database TIMER  (http://cistrome.org/TIMER/) was employed to identify the expression levels of SH3GL1 in a variety of tumor types. The results showed in (Fig 1A) revealed that SH3GL1 was markedly overexpressed in liver, lung, and breast cancer, suggesting that SH3GL1 may execute its function as a novel oncogenic driver in those cancer tumorigeneses. We further validated this finding using TCGA dataset and the analysis showed that SH3GL1 expression was significantly increased in liver cancer (Fig 1B). To further examine the relationship of SH3GL1 expression and liver cancer patients’ survival, we performed Kaplan-Meier survival analysis in TCGA dataset and the results revealed a statistically significant worse prognosis for liver cancer patients with high SH3GL1 expression levels compared with those with low levels roles of SH3GL1 (Fig 1C). Additionally, we next assessed expression pattern of SH3GL1 in clinical specimens of liver patients. Immunohistochemistry (IHC) staining assays were performed in a total of 60 liver tissue specimens and its corresponding normal tissues. As shown in (Fig 1D-E), SH3GL1 staining was negative or weak in para-tumor tissues and SH3GL1 expression level was significant high in the liver cancer. Importantly, we detected the expression of SH3GL1 in several HCC cell lines and normal hepatic cells LO2. As shown in (Fig 1F-G), comparing to LO2, both mRNA and protein expression of SH3GL1 were markedly overexpressed in HCC. Given that CD15 and CD133 have been widely accepted as liver CSCs biomarkers, CD15 and CD133 double positive cells could initiates more tumor spheres in HCC cell lines or HCC primary cells . We then sorted CD15+CD133+ cells, namely liver CSCs, using flow cytometry to initiate liver CSCs from Huh-7 and HepG2 cells. Actually, we found that the CD15+CD133+ cells form more tumor spheres than the CD15-CD133- (none CSCs) cells (data not shown). To further elucidate the biological role of SH3GL1 in CSCs maintenance, we compared the expression of SH3GL1 in liver CSCs and none CSCs and observed that SH3GL1 expression was markedly higher in liver CSCs than none CSCs (Fig 1H), implying that increased SH3GL1 expression may facilitate liver tumorigenesis. These data strongly support that the expression of SH3GL1 was substantially upregulated in liver cancer and associated with poor clinical outcomes of liver cancer patients.
SH3GL1 is essential for sustaining liver CSCs stemness and growth
To achieve a new avenue for the potential significance of SH3GL1 in liver tumorigenesis, we used short hairpin RNAs (shRNA) approach to directed ablate SH3GL1 expression in Huh-7 and HepG2 cells, which represent the highest expression of SH3GL1 (Fig 1F-G). The shRNA knockdown efficiency was examined (Fig 3C). The shRNA mediated depletion of SH3GL1 did not affect SH3GL2 and SH3GL3 expression (data not shown). Actually, SH3GL1 depletion remarkably impaired Huh-7 and HepG2 cell growth (Fig 2A). This finding was further confirmed by BrdU incorporation analysis. As shown in (Fig 2B-C), silencing SH3GL1 decreased Huh-7 and HepG2 proliferation rate, as reflected by the reduced proliferative cells in SH3GL1 silencing cells. Consistently, knocking down SH3GL1 inhibited Huh-7 and HepG2 colony formation ability (Fig 2D-E). As colony formation ability represents liver CSCs self-renewal maintenance capability, implying that SH3GL1 might executes crucial roles in sustaining liver CSCs stemness.
Tumor sphere formation assay was performed to investigate the functional role of SH3GL1 in liver CSCs maintenance. We observed that Huh-7 and HepG2 CSCs tumor sphere enrich ability was inhibited following silencing SH3GL1, as indicated by decreased tumor spheres number derived from liver CSCs following silencing SH3GL1 (Fig 2F-G). This observation was further confirmed by in vitro limiting dilution assay. As shown in (Fig 2H), targeting SH3GL1 inhibits liver CSCs self-renewal ability. All together, these data indicate that SH3GL1 plays critical roles in liver cancer cell proliferation and sustaining liver CSCs self-renewal.
SH3GL1 activates Wnt/b-catenin signaling
To explore the underlying mechanisms of SH3GL1 potentiates liver CSCs self-renewal, we detected two key downstream genes expression profiles of four key signaling pathways implicated in liver CSCs self-renewal, including Wnt/β-catenin , Notch , STAT3 , Hedgehog  signaling pathways. We found that c-Myc and CCND1, which are β-catenin downstream genes, were dramatically decreased following silencing SH3GL1 in Huh-7 and HepG2 cells (Fig 3A-B). On the contrary, silencing SH3GL1 did not affect the expression of Hedgehog, STAT3 and Notch downstream genes (Fig 3A-B), implying that SH3GL1 primes liver tumorigenesis through activating β-catenin signaling. Consequently, SH3GL1 disruption dramatically repressed the expression of β-catenin, c-Myc and CCND1 comparing with scrambled control shRNA (shCtrl) cells in liver CSCs SH3GL1 (Fig 3C). Notably, we explored the expression profile of SH3GL1 and c-Myc and CCND1 in the TCGA dataset and found that SH3GL1 was remarkably associated with c-Myc and CCND1 expression (Fig 3D).
To further investigate the underlying mechanisms that SH3GL1 upregulates β-catenin expression thereby promoting liver tumorigenesis, immunofluorescence assay was employed to determine whether there has any physically interaction of SH3GL1 and β-catenin in two independent HCC specimens. As shown in (Fig 3E), SH3GL1 was co-localized with β-catenin in two clinical HCC specimens (Fig 3F). This finding was further verified by Co-IP assay. As shown in (Fig 3G), SH3GL1 was found to interact with β-catenin in HepG2 CSCs. Immunofluorescence staining for β-catenin in HepG2 CSCs to characterize the biological significance of interaction between SH3GL1 and β-catenin. As shown in (Fig 3H), silencing SH3GL1 decreased total and nuclear part of β-catenin expression in HepG2 CSCs, suggesting that SH3GL1 promotes β-catenin signaling may specifically decrease β-catenin expression. Additionally, TOP-Flash reporter assay was employed to ascertain whether knockdown SH3GL1 affect β-catenin transcriptional activity. As shown in (Fig 3J), disruption SH3GL1 substantially repressed the activation of the TOP-Flash reporter in liver CSCs under the activation of Wnt3A condition. Consistently, knocking down SH3GL1 significantly decreased β-catenin expression (Fig 3K). Taken together, these data demonstrate that SH3GL1 directly and physically interacts with β-catenin, thereby activating β-catenin signaling to potentiate liver tumorigenesis.
Targeting SH3GL1 induces regression on HCC cell growth in vivo
We examined the effects of SH3GL1 silencing on the HCC growth in vivo using xenograft tumor growth model. A total of 2 X 106 Huh-7 cells expressing with SH3GL1 control shRNA or SH3GL1 shRNA were injected into the groin of mice. As expected, xenograft tumors derived from SH3GL1 shRNA cells grow significantly slower than control shRNA tumors (Fig. 4A-B) and tumor weight was also smaller than control tumors (Fig. 4C). Additionally, IHC staining was employed to examine the biological function of SH3GL1 on β-catenin activation. As shown in (Fig. 4D-E), β-catenin staining signal was dramatically decreased in xenograft tumors derived from SH3GL1 shRNA cells than control shRNA tumors. These data strongly support that β-catenin could be activated by SH3GL1 and SH3GL1 plays vital roles in liver tumorigenesis.