Understanding HCC (Hepatocellular Carcinoma) Development and Progression

Liver cancer is the fourth leading cause of cancer-related death worldwide, of which hepatocellular carcinoma (HCC) is the most common primary cancer accounting for 75-85% of cases. HCC can be classified into several histological subtypes, reflecting the heterogeneity of the disease. This heterogeneity, including the cancers’ cell plasticity, is known to be, in part, determined by the microenvironment.

The predominant cytokine signaling pathway for the development and progression of HCC cells, which connects the tumor cells with the microenvironment, is the TGF-β signaling pathway. The namesake of this pathway is a polypeptide belonging to the transforming growth factor beta superfamily of cytokines. Implicated in a number of functions, including cellular differentiation, proliferation and migration, TGF-β is capable of inhibiting hepatocyte proliferation and suppressing progenitor properties of hepatocyte-initiating cells. This is achieved by binding TGF-β type II receptor (TGFBR2) and activating TGF-β type I receptor (TGFBR1)-downstream Smad2/3 transcription factors.

The complexity of this disease is underscored by studies that have demonstrated how TGF-β1 signaling accelerates tumor progression by stimulating angiogenesis and promoting metastasis. Indeed, high TGF-β1 expression coupled with low TGFBR2 expression are predictive of poor outcomes in HCC. Moreover, the expression of TGFBR2 appears to be crucial in phenotypic shifting from TGF-β1-dependent proliferation inhibition to disease progression. The conditional functions of TGF-β1 reflect the intricacy of the TGF-β signaling pathway in HCC development and progression, and imply the need for sophisticated therapeutics. 

In addition, BMP proteins, which are also members of the TGF-β superfamily, can bind BMPR2 and BMPR1 on the cell surface and phosphorylating Smad1/5/8 downstream. The crosstalk between the TGF-β and BMP pathways has been the focus of much attention in the field of renal disease and bone formation with outcomes of these diseases relating to the balance between TFG-β and BMP signaling.

Building upon this information, a team of Chinese researchers led by Junya Ning, proposed that the imbalance between the TGF-β and BMP pathways is likely to be implicated in the progression of HCC. As current treatments for HCC fall short in preventing tumor reoccurrence and metastasis, thereby limiting long-term survival, there exists an imperative to devise novel strategies to combat the disease. Ning et al. set out to explore the interactive network between TGF-β and BMP pathways in HCCs by using the gene-expression profiles of 359 HCC samples provided by The Cancer Genome Atlas and 197 primary HCC tissues from Tianjin Medical University Cancer Institute & Hospital. Furthermore, they aimed to determine the core factors orchestrating the interaction between the TGF-β and BMP pathways, and identify the imbalance of TGF-β1/BMP-7 pathways in HCCs.

By observing RNA sequencing data extracted from HCC samples, they found that the imbalance between TGF-β1/BMP-7 pathways was detectable at the mRNA level, and was associated with poor clinical outcomes and aggressive pathological features. The significance of this imbalance was then evaluated at the protein level. This was achieved by comparing two cohorts. Cohort I comprised 64 patients who had each provided samples of primary HCC tissues, which then underwent immunohistochemistry staining to detect key proteins along the TGF-β1 pathway and BMP-7 pathway.

Overall survival was compared according to the expression of various proteins. It was observed that patients with specific protein profiles — low TGFBR2, high TGF-β1, high ACVR1, and high BMP-7 expression — had reduced overall survival compared to those in the alternative expression groups. They also found that TGFBR2 expression was significantly negatively correlated with ACVR1 expression, and samples expressing low TGFBR2 and high ACVR1 reflected worse clinical outcomes than the other patients.

Cohort II comprised 133 primary HCC tissue samples, which were used to detect expression patterns of TGFBR2 and ACVR2 in situ using immunofluorescence (IF) staining combined with multispectral imaging. This technique involved the washing and blocking of slides with antibodies followed by imaging using the TissueFAXS SPECTRA by TissueGnostics.

The TissueFAXS SPECTRA is a multispectral tissue cytometer based on a liquid crystal tunable filter (LCTF) and spectral unmixing engine of StrataQuest analysis software which enables the acquisition of higher numbers of fluorecscence channels for high content phenotyping in tissue sections. Permitting the separation of multiple colors the TissueFAXS SPECTRA allowed the liver slides to be imaged to reveal the DAPI staining, hepatocyte paraffin antigen 1, TGFBR2, and ACVR1. The autofluorescence was excluded within the process of spectral unmixing; the spectral signature of non-specific staining in the tissue (common in liver tissue) was detected and removed using the spectral unmixing engine and the multispectral data acquired by the LCTF in the form of a LAMBDA stack. Consistent with earlier findings, TGF-β1/BMP-7 pathway imbalance was validated at the protein level and linked to aggressive pathological features and poor clinical outcomes.

Through further experimentation, Ning et al. demonstrated that the imbalance of TGF-β1/BMP-7 pathways greatly increased HCC cell invasion — via the upregulation of the genes associated with epithelial-mesenchymal transition (EMT) and stemness — through stimulating the production of inhibitor of differentiation 1 (ID1). This imbalance was shown to be regulated by a microRNA (miR)-17-92 cluster, which promotes the imbalance of TGF-β1/BMP-7 pathways by interfering with TGFBR2 mRNA expression and enhancing ACVR1 protein expression via Smurf1 (a gene encoding an enzyme that acts as a negative regulator of the BMP signaling pathway) silencing.

It was then discovered that M2-polarized tumor-associated macrophages (M2-TAMs) affect the imbalance of TGF-β1/BMP-7 pathways in HCCs. By releasing extracellular vesicles containing the miR-17-92 cluster, M2-TAMs increase the level of clusters within HCC cells, thereby exacerbate the imbalance of the TGF-β1/BMP-7 pathway.

Finally, using mice xenografts, reversal of the pathway imbalance was shown to effectively attenuate M2-TAM-abundant HCC growth and metastasis in vivo. Altogether, this led Ning et al. to propose that imbalance between the TGF-β1/BMP-7 pathways is a feasible prognostic biomarker for HCC, and restoration of this balance might serve as a viable strategy to combat HCCs.

This study has significant implications in understanding HCC development and progression and opens the door to novel therapeutics to treat the disease. However, given the acute heterogeneity found within the tumor and microenvironment, in addition to the complex etiologic diversity of HCCs, Ning et al. highlight that patient-derived xenograft HCC models are required to validate the therapeutic efficacy of the TGF-β1/BMP-7 pathway imbalance. 

References and Further Reading

1. Ning, J., et al. (2021). Imbalance of TGF-β1/BMP-7 Pathways Induced by M2-polarized Macrophages Promotes Hepatocellular Carcinoma Aggressiveness. Molecular Therapy. 
2. Shi, C., et al. (2014). Incorporating spatial information in spectral unmixing: A review. Remote Sensing of Environment.

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