The leading cause of setbacks in clinical oncology practice pertinent to HCC is attributed to delay in early diagnosis alongside increased mortality rates. Delayed diagnosis is largely ascribed to the dearth in comprehensive knowledge with regard to the molecular mechanisms tangled in HCC developmental cascades. In order to identify the most suitable target specific drugs, advanced oncotherapeutic research demands a thorough information on polypharmacology, which enables exploration of multiple targets involved in disease pathogenesis and progression.
Phase-1: Target identification
Analysis of cross-talk gene involved in the development of Cirrhosis
Our study has predicted significant upregulation of STAT1 and MX1 during the changeover of normal liver to cirrhotic liver. This is supported by the fact that, STAT1, a crucial element in JAK/STAT signaling pathway is upregulated owing to activation of IFN β by HCV infected hepatocytes in due course of cirrhosis. Also, STAT1 is known for its tumor-suppressive actions in several types of cancer such as metastatic melanomas, colorectal, esophageal and pancreatic cancers. Conflicting with the aforementioned findings, STAT1 is also reported as a tumor promoter in breast carcinoma and soft tissue sarcoma, which further correlated with its poor prognosis and metastasis. [8]. Disregarding the existing controversies, we have validated STAT1 through GEPIA analysis. This has revealed no significant association of STAT1 in OS and DFS of HCC patients. This surprising result can be explained by the fact that, although STAT1 was identified as a substantial cross-talk gene in the development of cirrhosis, its impact was not vital enough in advanced neoplastic stages of HCC. The overexpression of STAT1 in cirrhosis owing to its extensive contemplation as a molecular target of anti-inflammatory therapies can be attributed to its involvement in chronic inflammatory conditions [9]. However, our research findings did not limit the role of STAT1 only to preneoplastic stages but also predicted its indirect involvement in interacting with other cross-talk genes such as MX1(C), CXCL11 (DN) and IGF1 (E).
MX1, a cross-talk gene which was upregulated at cirrhosis stage is an Interferon Stimulated Gene (ISG). It is reported to be overexpressed in HCV infected liver cells in chimpanzee models [10]. Similar to STAT1, MX1 was also not significantly associated with OS and DFS. However, our analysis visualized the interaction of MX1 with CXCL11, a cross-talk gene involved in the transition of C-DN. Though significant STAT1 and MX1 expression was not observed in later stages of HCC, their indirect involvement is suspected.
Scrutinizing the association of cross-talk genes in the progression of C-DN
We identified significant downregulation of six cross-talk genes such as CXCL11, CCL19, IL7R, CFTR, PROM1 and KRT19 in the evolution of C to DN.
CXCL11, a chemokine that regulates cytokine facilitated immune responses and angiogenesis, is recognized as a crucial biomarker in chronic HCV-associated cirrhosis. Upregulation of CXCL11 is associated with pancreatic cancer, whilst, silencing of the same was reported to inhibit tumor growth and metastasis in lung, bladder and colorectal cancers [11].
Herein, downregulation of CXCL11 in the transition of C to DN was seen, however, its expression was not discernible in further HCC stages. This gene showed a direct interaction with CCL19, a gene which was found to be involved in the development of DN. However, it did not exhibit direct communication with any of the cross-talk genes in E and A. Additionally, it showed insignificant association with OS and DFS [12].
CCL19, an inflammatory chemokine that regulates adaptive immunity is a prognostic biomarker in cervical and breast cancer. Controversial to the aforesaid statement, CCL19 therapy is contemplated to be beneficial in reducing the tumor burden in lung cancer [13]. Validation via GEPIA has proven an insignificant association of CCL19 with OS and DFS of HCC patients.
IL7R, an indispensable molecule allied with innate and adaptive immunity is deemed to be genetically amplified in various autoimmune disorders such as biliary cirrhosis, ankylosing spondylitis, multiple sclerosis and liver diseases in HIV-HCV co-infected patients [14]. Similarly, IL7R also causes activation of PI3-K and JAK-STAT signaling pathways which are reckoned for their underlying role in cancer progression [15]. Our study result emphasizes the downregulation of IL7R during the advancement of C to DN. This gene also lacks direct interaction with other HCC cross-talk genes and was not significantly associated with OS and DFS in HCC. However, it showed significant interaction with CFTR, an ATP-bound trans-membrane protein, which is renowned for its tumor suppressor activity. CFTR promoter methylation along with CFTR downregulation are reported as fundamental contributors to breast cancer, which in turn can be linked with poor patient survival [16].
Although, substantial downregulation of CFTR during the progression of C to DN was observed in our results, however, subsequent GEPIA analysis did not unveil remarkable association of CFTR in OS of HCC patients. Visualization of CFTR displayed interactions with IGF1, a cross-talk gene involved in the transition of preneoplastic to early HCC stages. Conversely, IGF1 did showed no interaction with any cross-talk genes involved in the progression of HCC.
PROM1, a cancer cell biomarker, ascertained to be frequently overexpressed in ovarian, esophageal and liver cancers was downregulated in the transition of C to DN [17]. GEPIA analysis also showed its poor association with OS and DFS in HCC.
KRT19 is another downregulated cross-talk gene which is widely distinguished for controlling cell-cycle arrest, apoptosis, invasion and metastasis in various cancers. It is substantiated to regulate HCC progression by microvascular invasion and HCC metastasis [18]. On the flip side, KRT19 was not significantly associated with OS and DFS in HCC.
Ultimately, none of the aforesaid downregulated cross-talk genes turned out to be significantly expressed or interactive in other stages of HCC beyond DN.
Decentering crucial cross-talk genes involved in transition of DN to E
Upregulation of HMMR and CCNB1 and downregualation of IGF1 were observed in the transition of DN to E.
HMMR that plays a central role in cell division by displaying peak action in G2/M phase, is a prognostic biomarker in different cancers. HMMR upregulation is inextricably linked to prostate, ovarian, colorectal and stomach cancers. Upregulation of HMMR in HCC patients reported by Lu et al (2020) was analogous to our study result [19]. The constructed PPI network also highlighted a significant interaction of HMMR with high node degree cross-talk genes in advanced HCC, thereby disinterring its extensive involvement in disease progression. OS and DFS of HCC patients revealed significant association of this gene in disease progression.
CCNB1, another upregulated cross-talk gene exerts its oncogenic activity in various cancers such as colorectal, pancreatic and gastric cancers [20]. In our study, this gene was found during the change-over from DN to A. OS and DFS in HCC showed strong association of CCNB1 in disease progression.
IGF1, a tumor promoter gene is related with cell proliferation and angiogenesis processes mediated via AKT and Ras pathways. This gene was downregulated in the progression of DN to E in our study. Evidences strongly suggests the crucial linkage of IGF1 with prostate, ovarian and colorectal cancers [21]. IGF1 was significantly associated with OS in HCC.
Unravelling key genetic factors underlying disease progression from E to A
Upregulation of pathological hallmarks of HCC such as CDK1, CDKN3, CDC20, KIF2C, ASPM, KIF20A, TPX2, KIF4A, TOP2A, BUB1, MAD2L1, CCNB1, BUB1B, CCNB2, PBK, NCAPG, NEK2 and CENPF were identified in the furtherance from E to A. Based on UALCAN database analysis, nine highest node degree cross-talk genes that were predominantly expressed in 371 HCC samples in comparison to 50 normal liver samples available in TCGA were narrowed down for further analysis. KEGG pathway indicated the involvement of aforementioned cross-talk genes in cell-cycle regulation and p53-signaling pathways.
CDK1, a cell-cycle dependent kinase forms complex with its protein partners, CCNB1 and CCNB2. These complexes act on spindle assembly checkpoints of G2/M Phase in mammalian cell-cycle [22] and thereby enhance chromosome condensation and breakdown of nuclear envelope. This complex was recognized to be upregulated in rhabdomyosarcoma, glioblastoma malignancies and HCC [23]. Parallel to the literature-based evidence, our study also unearthed a dominant correlation of CDK1, CCNB1 and CCNB2 in OS and DFS of HCC patients.
CDC20, an upregulated cross-talk gene exposed in our study is a coactivator of APC/C, which controls the initiation of mitotic exit from cell-cycle. Overexpression of CDC20 was associated with oral squamous cell carcinoma, gastric, colorectal and lung cancers [24]. In our validation process, among all identified cross-talk genes, CDC20 depicts higher significance as well as good interaction with all the nine high node degree cross-talk genes. Despite being a strong contributor, it portrays poor OS and DFS in HCC patients with higher HR. Therefore, CDC20 was considered as a potential druggable target against HCC progression. This finding is in concordance with other laboratory studies evaluating the antineoplastic potential of CDC20 inhibitors [25].
Another upregulated cross-talk gene, MAD2L1 acts on spindle assembly checkpoints and inhibits the binding of CDC20 to APC/C, thereby, preventing the onset of anaphase unless and until proper alignment of all chromosomes is seen in the metaphase plate. Dysregulation of MAD2L1 leads to chromosomal instability which paves a way to tumorigenesis [26]. This gene showed significant association with OS and DFS in HCC.
Upregulated NEK2 plays a crucial role in kinetochore attachment, centrosome duplication and separation in S/G2 phase of cell-cycle. Numerous studies have reported the overexpression of NEK2 in HCC, leukemia, melanoma, bladder and breast cancers [27]. Herein, upregulated NEK2 alongside exhibiting considerable interactions with all the other high node degree cross-talk genes, was associated with poor prognosis of HCC patients as evinced through GEPIA analysis.
Upregulated TOP2A is a critical cross-talk gene in the carcinogenic process which plays a crucial role in DNA replication and transcription. It is reported for its strong alliance in a wide range of cancers such as ovarian, breast and endometrial cancers. Similar to our findings, Ma et al. reported the co-expression of kinesin superfamily (KIFs such as KIF20A, KIF4A, KIF2C), TPX2 and CENPF with TOP2A in papillary renal cell carcinoma [28]. Upregulated KIFs, TPX2 and CENPF are extensively involved in the breast cancer progression [29]. Our GEPIA analysis revealed that TOP2A, KIFs, TPX2 and CENPF affected the OS and DFS in HCC.
PBK is a serine-threonine kinase frequently found to be upregulated in cervical and prostate cancers. It increases cell proliferation via ERK pathway, alongside impairing death of cancer cells by apoptosis mechanisms. PBK overexpression promotes metastasis of HCC via activation of ETV4-uPAR signaling pathway [30]. In line with the above results, our study also highlighted the negative impact of upregulated PBK in survival analysis of HCC patients.
NCAPG is a mitosis-related chromosome condensation protein, which has been reported as an important prognostic marker and therapeutic target for HCC. The knockdown of NCAPG expression inhibited tumor cell growth and proliferation of HCC cell lines [31]. Parallel to this finding, our study showed that upregulation of NCAPG significantly affected the OS and DFS in HCC.
MELK, a prognostic biomarker of breast and ovarian cancer is implicated in the regulation of tumorigenesis. It is overexpressed in HCC with poor survival rate [32]. MELK regulates NF-kB pathway, a vital signaling pathway that is dysregulated in development and progression of cancer. Our study also showed upregulation of MELK with poor survival rate.
Overall analysis revealed CDC20 as a druggable target based on its interaction with other crucial cross-talk genes in HCC progression, impact on poor survival probability with high HR. Also, CDC20 inhibitors such as Delanzomib, Oprozomib and Marizomib are already shelved in trial against different cancers [33]. Hence, CDC20 is considered for phase-2 computational simulation studies.
Phase-2: Computational Simulation Studies to explore potential drugs against CDC20
Molecular docking technique revealed Labetalol and Deferasirox as potential repurposable drugs against CDC20. Labetalol was proved to exert its anticancer activity in combination with Metformin against breast cancer [34]. Similarly, Deferasirox was proved to exhibit significant antineoplastic effect in oesophageal cancer [35] and multiple myeloma [36]. As Labetalol and Deferasirox were not reported for HCC, they were further examined for their dynamic behaviour through MD simulations. Deferasirox-CDC20 fluctuated out of the range throughout the simulation, whereas, Labetalol-CDC20 exhibited no deviation from the protein backbone and showed better stable interaction. Hence, Labetalol was identified as a repurposable drug against HCC. To validate this virtual findings, cytotoxicity of Labetalol was investigated through MTT assay.
Phase-3: In-vitro experimental validation
MTT assay is a sensitive method to confirm the cytotoxicity of anticancer agents prior to preclinical evaluation. Herein, Labetalol significantly decreased cell growth in HepG2 cell line with an IC50 of 200.29µg/ml indicating its potential cytotoxicity against HCC.