Follistatin Like 5 (FSTL5) Inhibits Epithelial to Mesenchymal Transition in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common malignancy globally and the third leading cause of cancer-related mortality. Despite advancements in treatment, postoperative recurrence and metastasis remain significant challenges, driven largely by epithelial to mesenchymal transition (EMT). EMT facilitates tumor cell detachment, migration, and dissemination by downregulating epithelial markers like E-cadherin and upregulating mesenchymal markers such as vimentin. The Follistatin-like (FSTL) protein family, which regulates cell differentiation and organ development, has emerged as a potential therapeutic target in cancer. Follistatin Like 5 (FSTL5), a member of this family, shares structural homology with Follistatin (FST), a known inhibitor of activin and TGF-β signaling. This study investigates the role of FSTL5 in HCC progression, EMT regulation, and its clinical relevance.

Clinical Relevance of FSTL5 in HCC

The study analyzed 321 pairs of HCC and adjacent non-tumor tissues using immunohistochemistry (IHC). FSTL5 expression was significantly lower in HCC tissues (9.97%) compared to paracancerous tissues (82.55%) (( chi^2 = 340.15 ), ( P 200 , mu g/L ): 25 vs. 205, ( chi^2 = 24.36 )), and better liver function (Child-Pugh grade A: 27 vs. 171, ( chi^2 = 5.21 )).

FSTL5 Modulates EMT Markers in HCC

IHC analysis demonstrated a strong positive correlation between FSTL5 and E-cadherin (( r = 0.38 ), ( P < 0.001 )) and a negative correlation with vimentin (( r = -0.385 ), ( P < 0.001 )) in HCC tissues. To validate these findings, SK-Hep1 and MHCC-LM3 HCC cell lines were transfected with lentiviral vectors overexpressing FSTL5 (Lv-FSTL5). Quantitative real-time PCR and Western blotting confirmed that FSTL5 overexpression upregulated E-cadherin mRNA and protein levels while suppressing vimentin. In SK-Hep1 cells, E-cadherin mRNA increased (( t = 45.03 ), ( P < 0.001 )), and vimentin decreased (( t = 67 ), ( P < 0.001 )). Similar results were observed in MHCC-LM3 cells (( t = 50 ) for E-cadherin, ( t = 72.75 ) for vimentin, ( P < 0.001 )).

FSTL5 Suppresses HCC Cell Proliferation and Invasion

Cell proliferation was assessed using the CCK-8 assay. Overexpression of FSTL5 significantly inhibited growth in both SK-Hep1 and MHCC-LM3 cells. For SK-Hep1, proliferation rates decreased at day 3 (( t = 7.324 ), ( P = 0.018 )), day 4 (( t = 6.23 ), ( P = 0.021 )), and day 5 (( t = 10.21 ), ( P = 0.003 )). In MHCC-LM3, reductions were observed at day 3 (( t = 4.32 ), ( P = 0.037 )), day 4 (( t = 7.49 ), ( P = 0.012 )), and day 5 (( t = 9.37 ), ( P = 0.009 )).

Transwell invasion assays further revealed that FSTL5 overexpression reduced invasiveness. SK-Hep1 cells showed a 63% reduction in invaded cells (( t = 21.57 ), ( P < 0.001 )), while MHCC-LM3 exhibited a 58% decrease (( t = 18.04 ), ( P < 0.001 )). These results highlight FSTL5 as a suppressor of both proliferative and invasive capacities in HCC.

Mechanistic Insights and Therapeutic Implications

FSTL5’s role in EMT inhibition aligns with prior studies on FST family proteins. FST directly binds TGF-β3 to suppress EMT in mammary cells and inhibits EMT-associated transcription factors (Snail, Zeb, Twist1) in ovarian models. The study posits that FSTL5 may regulate EMT through pathways involving Wnt/β-catenin or Yes-associated protein (YAP). YAP, a known EMT promoter, is upregulated in HCC and interacts with transcriptional regulators of vimentin and slug. FSTL5’s ability to inhibit YAP activation could explain its anti-EMT effects, though further mechanistic studies are warranted.

Clinically, the correlation between FSTL5 downregulation and aggressive HCC phenotypes suggests its utility as a biomarker for metastasis risk and prognosis. Restoring FSTL5 expression via gene therapy or small-molecule activators could offer novel therapeutic strategies to curb EMT-driven recurrence.

Limitations and Future Directions

The study’s retrospective design and reliance on a single cohort (( n = 321 )) necessitate validation in larger, multi-center populations. While in vitro models confirmed FSTL5’s functional roles, in vivo experiments are needed to assess its efficacy in animal models. Additionally, the precise molecular mechanisms linking FSTL5 to EMT regulators like YAP or Wnt/β-catenin require further exploration.

Conclusion

FSTL5 acts as a critical suppressor of EMT in HCC by modulating E-cadherin and vimentin expression. Its downregulation correlates with advanced tumor stages, metastasis, and poor survival, underscoring its potential as a prognostic marker and therapeutic target. Strategies to enhance FSTL5 activity may improve outcomes in HCC patients by mitigating EMT-driven progression.

DOI: doi.org/10.1097/CM9.0000000000000847

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