Deciphering the Ras/MAPK Signaling Pathway in the Progression and Treatment of Hepatocellular Carcinoma

Hepatocellular Carcinoma (HCC) is the most prevalent liver tumor affecting 85-90% of all patients [1]. The significant reason for HCC is cirrhosis, it affects the majority of patients [2]. It remains a significant public health issue due to its status as the second primary reason of cancer-related death and the fifth greatest prevalent cancer globally [3]. The occurrence of HCC is increasing fastly in emerging countries due to various reasons i.e., the rising prevalence of cirrhosis from numerous causes specifically Nonalcoholic Steatohepatitis (NASH), an increase in the survival rate of cirrhotic patients but steadily rising the risk of Hepatitis C Virus (HCV) infection and obesity [4]. Persons with chronic HBV or HCV infections, particularly those with liver cirrhosis, have a significantly extreme risk of HCC contrast to healthy people [5,6]. All of these risk factors contribute to persistent inflammation, which promotes hepatic fibrosis, hepatic cirrhosis, and ultimately HCC [7].

The multi-step process of HCC progression is controlled by the epigenetic and genetic changes to deactivate tumor suppressor genes and activate oncogenes therefore dysregulating the essential cellular functions. HCC is inherently diverse tumor and various molecular processes influence its growth [8,9]. The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/ mTOR), Janus Kinase/Signal Transducer Activator of Transcription factor (JAK/STAT), Hippo signaling pathways, p53 pathway, Hedgehog (HH), and the Wnt/β-catenin, are the major pathways associated to HCC [10]. The primary mechanism of liver cancer has notably the Ras/Raf pathway activation been identified as the deregulation of cell proliferation [11]. Several studies revealed the Ras/MAPK pathway is a crucial biological route involved in the progression of liver tumors in both preclinical as well as in humans [12]. It is reported that Ras/MAPK pathway is the most important in developing HCC among the other signaling pathways [13]. This route is activated in 50-100% of HCC patients as per studies [14]. The HBx and HCV core proteins mediate the initiation of the Ras/MAPK pathway to cause heptocarcinogenesis [15]. Sirtuin 1 (SIRT1) plays a critical role in HCC by promoting tumorigenicity through regulation of oncogenes, metabolic reprogramming, and enhancing cell survival [16,17]. SIRT1 modulates MAPK signaling, which is pivotal for cell proliferation, differentiation, and survival. By deacetylating key components of the MAPK pathway, SIRT1 can enhance tumorigenicity, promote metastasis through epithelialmesenchymal transition, and contribute to chemoresistance. Targeting SIRT1-MAPK interactions presents a promising strategy for improving HCC treatment outcomes [18]. SIRT1 activators and inhibitors can significantly impact the effectiveness of drugs targeting the Ras/MAPK pathway in HCC by modulating cell proliferation and survival mechanisms, potentially enhancing or diminishing therapeutic outcomes [19]. This review reports the molecular cascades to activate the Ras/MAPK pathway in HCC and molecular inhibitors to block Ras/MAPK signaling cascades for treatment. Additionally, suggest current and effective treatment strategies to focus on the Ras/MAPK signaling pathway. The phosphorylation of MAPK and the expression of the proteins c-fos and c-jun reveal to determine the role of the signal transduction cascade in the development of hepatocarcinogenesis.

Members of the multigene families Ras, Raf, and MEK include the three members Ha-Ras, N, and Ki of Ras, the three members A, B and Raf1 of Raf, and the five members of the MEK gene family (MEK1-5) are responsible to regulate Ras/MAPK pathway [20]. The MAPK cascade are serine/threonine kinases change the extracellular molecules such as hormones and growth factors into intracellular signals to regulate differentiation, survival, and proliferation of cells [21]. G-Protein-Coupled Receptor (GPCR) or Receptor Tyrosine Kinase (RTK) mediates signaling from cell surface receptors to the nucleus is thought to activate cellular processes [22]. The superfamily of Ras/MAPK pathway consists of some members based on the degree of similarity and makes up seven groups: Extracellular signal-Regulated Kinase (ERK) 1/2, ERK ¾, ERK5, ERK 7/8, Nemo-Like Kinases (NLKs), p38 a/b/c/d and Jun N-terminal JNK 1/2/3 kinases [23]. Normally, the Ras/ MAPK pathways interact with other pathways including the PI3K/ Akt/mTOR, Rho/actin, Wnt/β-catenin, and TGFβ/Smads to regulate cellular functions [24].

The Ras protein is a GTPase protein and four isoforms expressed by three distinct genes; H, N, and K-Ras4A/4B [25]. Ras is the initial ERK 1/2 intracellular effector and activated by different extracellular stimuli as RTK’s activation through different growth factors [26]. The stimuli switch the state of Ras from an inactive GDP bound form to GTP active form therefore trigger the various pathways associated with cell survival, growth, differentiation, and migration via means of several effectors [27]. GTP bound Ras activates the Raf-1 kinase, which further drives it to the cell membrane. Raf-1 activation phosphorylates the MEK 1/2 and subsequently phosphorylates ERK 1/2 accordingly, a number of proteins control the Ras/MAPK pathway. Activated ERK moves to the nucleus and stimulates several TF, the AP-1 family members as c-Fos and c-Jun [28]. These factors transcribe genes involved in cells progression or other biological activities by bind to promoter region specifically on AP-1 binding sites [29].

Guanine Nucleotide Exchange Factors (GEFs) and GTPase- Activating Proteins (GAPs) regulate the activation of Ras. GEFs convert GDP to GTP although GAPs hydrolyse the GTP-Ras [30]. Antiapoptotic and Ras proliferative Signals are regulated by members of the Ras Association domain Family (RASSF) which consists of NORE1A, NORE1B, RASSF1A, RASSF1C, RASSF2-10 [31]. The Spred family protein prevents Raf activation, and the Raf kinase inhibitor protein (RKIP) prevents MEK from phosphorylation by Raf-1, so are further controllers of the Ras/MAPK system [28]. Sprouty proteins control the Ras/MAPK pathway either by blocking Raf or the growth factor receptor bound-2 (GRB-2)-SOS complex [28]. Ras not only activates Raf and the MAPK pathway but a different additional effectors interact with Ras through the Ras-Binding Domain (RBD) [32]. The primary ones are the Phospholipase C epsilon (PLCe), Ral Guanine Nucleotide Dissociation Stimulator (RalGDS), and PI3K [33]. Ras stimulates these effectors to activate a number of pathways including the PI3K pathway involved in survival, growth, proliferation, angiogenesis, also the control of glucose and lipid metabolism [34]. The intracellular mechanisms linked to the regulation of MAPK pathway in HCC were addressed in more detail in behind paragraphs and shortly discussed the influence of crosstalk involving Ras with other pathways as well as the function of certain other Ras effectors in cancer development (Figure 1).

Figure 1:Schematic representation of MAPK/ERK signaling pathway and its regulation. MAPK/ERK signaling pathway is initiated by the binding of growth factors to receptor tyrosine kinases (RTKs) on the cell surface. Activation of RTKs leads to the recruitment of the adaptor protein GRB2 and the guanine nucleotide exchange factor SOS, which facilitate the exchange of GDP for GTP on Ras, activating it. Active Ras (Ras-GTP) then activates RAF, which in turn phosphorylates and activates MEK1/2. MEK1/2 phosphorylates ERK1/2, leading to its activation. Activated ERK1/2 translocate to the nucleus, where it phosphorylates transcription factors such as C-Fos and C-Jun, promoting gene transcription. The pathway is negatively regulated by several mechanisms: Sprouty inhibits GRB2 and RAF; Spred inhibits RAF; RASSF promotes the hydrolysis of GTP to GDP on Ras, inactivating it; RKIP inhibits MEK1/2; and DUSP1 dephosphorylates ERK1/2, inactivating it. These regulatory mechanisms ensure precise control over cell signaling events.

Current considerations on signaling pathway in hepatocarcinoma

Ras/MAPK pathway dysregulation is linked to cancerous transformation and development, including liver cancer as it performs a decisive role to regulate the important cellular functions such as the survival and proliferation of cells [35]. Liver cancer exhibits higher levels of MEK 1/2 and ERK 1/2 expression than the surrounding nonneoplastic liver tissue [36]. Additionally, a strong relation between expression of MAPK and c-Fos protein, c-Fos and the cyclin D1, MAPK and cyclin D1 is found in human HCC [37]. The MAPK pathway is implicated in tumor growth where the therapy of HCC cells with a particular Ras/MAPK inhibitor suppresses the tumor growth and causes apoptosis [38].

Ras mutations

The Ras gene mutations reported about 20-30% in all human tumors and a high incidence in the colon (40-50%), pancreas (57%), salivary gland (15%) and skin cancer (17%) [39]. The point mutations linked to cancer are in Ras protooncogene primarily affect codons 12, 13 and 61 [40]. These mutations make the Ras protein active, allowing it to trigger downstream processes [41].

Ras GTPase-activating proteins

The Ras family consists of 150 members and are usually categorized into Ras, Ran, Rho, Rab, and Arf families. Ras GAPs convert the Ras-GTP activated form to the GDP-inactivated by increasing the Ras GTPase activity, this results to convert the GTP to GDP. Inhibiting GAP activity triggers accumulation of the activated Ras, allowing it to activate downstream effectors [42]. RAS GAPs are categorized into a number of different families, such as RASA1 or p120GAP, GAP1 proteins (RASAL1, RASA2-4), NeuroFibromin (NF1), the SynGAPS (RASAL2/3, SynGAP1, DAB2IP A/B), and the IQGAP family (IQGAP1-3) [43]. Ras GAPS family members like RASAL1, DAB2IP and hDAB2IPA are persistently downregulated in a number of human HCC cell lines including HepG2, SK-Hep-1, Hep 3B, PLC/PRF/5 probably with hypermethylation in the promoter [28]. DAB2IP, RASAL1, and NF1 expression levels were significantly suppressed in patients and was primarily due to promoter hypermethylation [44].

The siRNA-mediated inhibition of RASAL1, PITX1 (a RASAL1 upstream inducer) and DAB2IP expression results in improved cell survival, partially as a result of increased RAS GEF activity [45]. The re-expression of RASAL1, PITX1 and DAB2IP results in a substantial decrease proliferation and an increased apoptosis in HCC cells [46]. Several GAPs can stimulate apoptosis in cancer cells to defend the individual. The five GAPs are Deleted in Liver Cancer 1 (DLC1), Regulator of G-protein Signaling 3 (RGS3), DOC-2/DAB2 interacting protein (DAB2IP), p120 RasGAP (RASA1) and STARD13 affect the number of pro-apoptotic and anti-apoptotic proteins or the associated signaling pathway lead to apoptosis [47]. The hDAB2IPA expression is discovered to be much lower in HCC with promoter hypermethylation found in >80% of liver tumors. DAB2IP downregulation is linked to a poor overall survival. The gene and protein expression of IQGAP1 is highly expressed in 84% of tumors whereas IQGAP2 are significantly suppressed in 78% of patients hence IQGAP1 has an oncogenic role and IQGAP2 maintains a tumor suppressor function [48] (Table 1).

Table 1:An overview of key components related to Ras signaling pathway regulation and downstream effects in the context of HCC.

RASSF1A and NORE1A are the two key players activate the Macrophage Stimulating 1 (MST1) kinase and BAX to cause both caspase dependent and caspase-independent apoptosis after coupled to Ras. RASSF1A and NORE1A must heterodimerize to start Ras-associated pro-apoptotic proteins [49]. The RASSF1A binds to Plasma Membrane Calcium Pump (PMCA) 4 proteins to inhibit MAPK pathway [50]. The function of inhibitors in proliferation describes readily why their dysregulation significantly contribute to the Ras/ MAPK pathway initiation in a variety of malignancies. RASSF1A and NORE1A typically exhibit promoter hypermethylation in many types of human malignancies [51]. The promoter hypermethylation is thought to be the primary mechanism for RASSF1A silencing, although post-translational mechanisms involving downregulation of microRNA also contribute to RASSF1A inactivation in HCC [52]. The serum DNA from HCC patients show irregular methylation of the RASSF1A gene promoter in patients indicating this genetic modification eventually used for early diagnosis in individuals with a significant risk to develop liver cancer [53]. The human HCC about 62% exhibit epigenetic inactivation of NORE1B [51] and an additional RASSF1A silencing is seen in 50-92% of these cases [54]. HCC samples do not exhibit any hypermethylation of the RASSF1C promoter [55] while RASSF4 may occasionally have abnormal epigenetic silencing [56].

Spread family of MAPK inhibitors

Sprouty-Related Protein EVH1 Domain (SPRED) interacts with Ras to prevent activation of Raf and translocate to the cell membrane, consequently acting as a negative mediator of the Ras/ MAPK pathway and acts as a tumor suppressor [57]. The SPRED family consists of four members in mammals: SPRED1-3 and EVE- 3. Spred1 is mostly expressed in embryonic tissues and adult brain and less in other tissues; While Spred2 is frequently present in adult tissues and is hardly ever found in embryonic tissues; Spred3 is in the liver; EVE-3 in the liver and the brain [58]. The SPRED expression reduces the ERK phosphorylation and suppresses tumor cell proliferation [59]. Spred-2 overexpression in HCC cell line SMMC-7721 causes apoptosis of cells via downregulation of anti-apoptotic protein Mcl-1 and the caspase pathway [58]. Finally, reduced Spred expression is linked to serious HCC as intrahepatic metastases and tumor proliferation [28].

SPRED proteins can no more prevent ERK activation upon the removal of EVH1 region [60]. The EVH1 domain target proteins at certain locations for action by specifically binding to prolinerich sequences involved in processes like synaptic transmission, skeletal control, cytoskeletal remodeling and cell proliferation [58]. The SPR domain has multiple roles in different SPRED proteins [61]. SPR deletions in the proteins SPRED1 and EVE-3 can still suppress ERK activation, whereas a comparable mutation in SPRED2 cannot [62]. The SPR domain of SPRED1 and SPRED2 encourages the development of heterodimers, which can respond to various stimuli in a temporal and spatial manner to carry out specific inhibitory functions [63]. SPRED3 has a lesser inhibitory activity than SPRED1 and SPRED2 but can still prevent ERK from being activated [64]. This shows the KBD domain is missing from SPRED3 that involve to suppress ERK activation. The KBD domain is thought to be necessary for SPRED1 and 2 phosphorylation and its existence improves the SPRED function [58].

Sprouty family of MAPK inhibitors

The Sprouty family of signal transduction protein contains 4 regulators i.e. Platelet-Derived Growth Factor Receptor (PDGFR-β), Vascular Endothelial Growth Factor Receptor (VEGFR), Fibroblast Growth Factor Receptor (FGFR) and Epidermal Growth Factor Receptor (EGFR) to change downstream cellular responses caused by GPCR and RTK [65]. Sprouty proteins primarily acting as modulators of the Ras/MAPK pathway and its dysregulation has linked to the metastasis and progression of many cancer types including lung, breast, colon and prostate cancer [66]. Sprouty homologues are differently expressed in HCC [28]. The expression of Sprouty 2 prevents ERK activation and HGF-induced proliferation [67] and the downregulation of Sprouty 2 promote cell proliferation hence the Sprouty family received the most attention in the field of HCC as a result of unrestricted activity of Ras/Raf pathway [68]. Moreover, the MAPK pathway is quickly activated by Sprouty 2 inactivation in association with AKT to cause hepatocarcinogenesis [28]. The downregulation of sprouty 2 is more significant in HCC with a poor prognosis [69].

Raf kinase

The Raf kinase is a downstream regulator of Ras molecule [70]. The three Raf kinases A-Raf, Raf-1, and B-Raf all are upstream regulators of the Ras/MAPK pathway [71]. The more cancercausing isoform, the B-Raf gene is mutated in many human cancers including 66% of skin cancers and a lower rate in papillary, colon, thyroid, and ovarian cancer [39]. The majority of B-Raf gene mutations (almost 90%) change valine to glutamic acid at position 599 (V599E), thereby increasing the kinase activity and triggers the activation of the MAPK cascades. Raf-1 mutations are extremely uncommon while A-Raf mutations have never reported [72].

This is in accordance with the evidence that prolonged expression of c-Raf-1 and Raf-1 play a carcinogenic effect in a number of tumors [73]. The RKIP is yet another element contributing to Raf-associated hepatocarcinogenesis. RKIP is a protein to blocks the activity of the Ras/MAPK pathway for cellular proliferation and progression [74]. Raf-MEK complexes interrupt up upon binding of RKIP to either Raf-1 or MEK under vigorous conditions to prevent downstream signaling of MEK [75]. Raf is improperly activated whenever RKIP-mediated inhibition of Raf is disrupted, it leads to upregulate MAPK pathway [76]. Human prostate, ovarian, breast, melanoma, and colorectal cancers have linked to reduced RKIP activity [77]. RKIP downregulation has been demonstrated in liver cancer and helps to activate the MAPK pathway [78]. The epigenetic transcription and promoter methylation within the RKIP coding sequence are the two primary factors causing the reduction of RKIP expression in cancers [76]. The RKIP protein expression is downregulated in HCC tissue in contrast to noncancerous liver samples [28].

MEK/ERK proteins

ERK 1/2 are the chief substrates of MEK 1/2 [79]. Activated ERK1/2 activates cytosolic and nuclear substrates such as Transcription factor AP-1 perform a fundamental role in regulating the survival and proliferation of cells [80]. Hence the high ERK activity has been seen in a different cell lines and tumors as lung, pancreas, kidney, colon, and ovary cancer [81]. The main isoform associated with normal hepatocyte proliferation is ERK ½ [82]. The MEK/ERK pathway activated by a number of mechanisms to favour the advancement of liver cancer by enhancing cellular survival, proliferation, tumor growth, cell motility, invasiveness, and angiogenesis [83]. The frequency of ERK activation affects how specifically ERK and DUSP1 interact [84]. The initiation of ERK 1/2 dysregulates the DUSP1 through ubiquitin-mediated proteasome degradation pathway to degrades DUSP1 and activate ERK 1/2 for prolonged period [85].

It is stated in the introduction, the Ras/MAPK pathway frequently interacts with other signalling pathways to regulate cellular processes both in healthy and in pathological conditions like liver cancer [10]. The Ras protein to activate Raf, a number of additional signaling molecules such as the PI3K, TIAM1, RalGDS and the PLCe proteins is activated to the interaction across pathways [86]. Moreover, TIAM1 stimulates cancer metastasis and is associated with poor prognosis in HCC patients [87]. The MAPK and PI3K/Akt/mTOR pathways interact at different levels which can have either a positive or negative effect on one another. Both mechanisms collaborate to encourage the survival and development of tumor cells, and favours carcinogenesis [88]. Consequently, the Ras/Raf, Wnt/β-Catenin, Rho/actin, and TGFb/Smads pathways make up a complicated network of interactions to promote tumor cell’s differentiation, proliferation, and tumor invasion [89].

The field of molecular therapeutics in primary liver cancer treatment was introduced by the various effectors of MAPK pathway, which include several therapeutic targets due to their significance in hepatocarcinogenesis. Sorafenib is a strong multikinase inhibitor primarily inhibits Raf kinases, PDGFR-α, VEGFR-2/3, and c-kit and is the initial systemic treatment authorized for advanced HCCs [90]. A variety of Ras/MAPK pathway inhibitors have developed over the past few years, and at the meantime, specific inhibitors for many of the pathway’s essential elements are being defined [28]. Small compounds like lonafarnib or tipifarnib block the MAPK pathway in an efficient manner. These compounds prevent the farnesyltransferase enzyme from catalyzing Ras protein farnesylation, a critical step that binds Ras to the cell membrane [91]. Regorafenib (SCH66336), a multikinase inhibitor, blocks BRAF, PDGFR-β, VEGFR-2/3, CSF- 1R, RET, c-Kit, and Flt3 [92]. Clinical trials, systemic drugs, and a number of widely recognized immunotherapy approaches all contribute to the advancement of innovative drug development. The Food and Drug Administration (FDA) approved a number of drugs between 2017 and 2020 for the treatment of HCC including immune checkpoint inhaibitors (pembrolizumab, nivolumab, durvalumab, tremelimumab, ipilimumab), multikinase inhibitors (sorafenib, lenvatinib, cabozantinib, ramucirumab, regorafenib and atezolizumab in combination with bevacizumab) [93]. The FDA for the treatment of HCC expressly approved multiple immune checkpoint inhibitors and antiangiogenic therapies and numerous other medications are now being tested in clinical studies. First, second, and third-line therapy has significantly changed the course of HCC with a cure rate of over 90%, nevertheless few studies have reported a concerning increase in the recurrence of HCC. The most frequently changed mediators (the TERT promoter, CTNNB1, and TP53) and dysregulated pathways (Ras/MAPK, β-catenin/Wnt, and PI3K/mTOR) associated with HCC have all been identified using molecular characterizations. The right anti-tumor activity and toxicity need to be balanced through the use of innovative drugs and therapies. A number of drugs are currently available to prolong their lives for individuals with advanced HCC. It is still unclear which HCC patients respond best to a certain drug and what the ideal therapeutic strategy (Table 2).

Table 2:A comprehensive overview of various drugs used in the treatment of hepatocellular carcinoma (HCC).

The future perspectives for this review are multifaceted and promising. As the understanding of hepatocarcinogenesis and the function of the Ras/MAPK pathway expands, individualized therapy based on the particular molecular patterns of each patient may become a reality. Early diagnosis and prognosis may be improved by the identification of certain biomarkers linked to Ras/MAPK pathway dysregulation in HCC. Novel therapeutic drugs that target this pathway could appear, possibly developing therapy approaches. The study of immunological responses and Ras/MAPK deregulation may lead to the development of new immunotherapies. The validation and improvement of Ras/ MAPK-targeted drugs depends on clinical trials, translational research, and ongoing monitoring of patient outcome. A more thorough knowledge of HCC will be possible with the integration of multi-omics data and the encouragement of multidisciplinary cooperation. Additionally, addressing the expanding problem of HCC on a global scale requires taking into consideration the effects on global health and establishing ways for early identification and universal availability of current drugs.

A thorough understanding of the abnormal molecular mechanisms leading to carcinogenesis will feasibly allow to predict the patient’s response to targeted therapy which might have a significant influence on clinical decision-making. Hepatocarcinogenesis, a multistep process results in the genetic and epigenetic change to activate oncogene, and inactivate tumor suppressor gene followed by changes in different signalling cascades. A number of intracellular processes in HCC like Ras/MAPK pathway is important for the growth and progression of HCC are dysregulated and lead to survival, differentiation, and proliferation of cells. The Ras/MAPK is the most effective signalling pathway that manages malignancy and controls apoptosis in cancer. The upregulation of the Ras/MAPK pathway has largely changed these cellular processes. This review discussed the several modulators to regulate the Ras/MAPK.

1. Zhou J, Sun HC, Wang Z, Cong WM, Wang JH, et al. (2018) Guidelines for diagnosis and treatment of primary liver cancer in China (2017 Edition). Liver cancer 7(3): 235-260.
2. Tarao K, Nozaki A, Ikeda T, Sato A, Komatsu H, et al. (2019) Real impact of liver cirrhosis on the development of hepatocellular carcinoma in various liver diseases-meta‐analytic assessment. Cancer medicine 8(3): 1054-1065.
3. Konyn P, Ahmed A, Kim D (2021) Current epidemiology in hepatocellular carcinoma. Expert Rev Gastroenterol Hepatol 15(11): 1295-1307.
4. Huang DQ, El-Serag HB, Loomba R (2021) Global epidemiology of NAFLD-related HCC: Trends, predictions, risk factors and prevention. Nature Reviews Gastroenterology & Hepatology 18(4): 223-238.
5. Kanwal F, Khaderi S, Singal AG, Marrero JA, Loo N, et al. (2023) Risk factors for HCC in contemporary cohorts of patients with cirrhosis. Hepatology 77(3): 997-1005.
6. Mushtaq H, Zarlashat Y, Ambreen A, Mujahid M, Kausar S, et al. (2024) Reviewing advances in understanding and targeting the MAPK signaling pathway in hepatocellular carcinoma progression and therapeutics. Agrobiological Records 15: 103-116.
7. Berumen J, Baglieri J, Kisseleva T, Mekeel K (2021) Liver fibrosis: Pathophysiology and clinical implications. WIREs Mechanisms of Disease 13(1): e1499.
8. Craig AJ, Von Felden J, Garcia-Lezana T, Sarcognato S, Villanueva A (2020) Tumour evolution in hepatocellular carcinoma. Nature Reviews Gastroenterology & Hepatology 17(3): 139-152.
9. Zarlashat Y, Abbas S, Ghaffar A (2024) Hepatocellular carcinoma: Beyond the border of advanced stage therapy. Cancers (Basel) 16(11): 2034.
10. Garcia-Lezana T, Lopez-Canovas JL, Villanueva A (2021) Signaling pathways in hepatocellular carcinoma. Adv Cancer Res 149: 63-101.
11. Alqahtani A, Khan Z, Alloghbi A, Said Ahmed TS, Ashraf M, et al. (2019) Hepatocellular carcinoma: Molecular mechanisms and targeted therapies. Medicina 55(9): 526.
12. Cui J, Shen HM, Lim LHK (2020) The role of autophagy in liver cancer: Crosstalk in signaling pathways and potential therapeutic targets. Pharmaceuticals 13(12): 432.
13. Yang S, Liu G (2017) Targeting the Ras/Raf/MEK/ERK pathway in hepatocellular carcinoma. Oncol Lett 13(3): 1041-1047.
14. Sun L, Xi S, Zhou Z, Zhang F, Hu P, et al. (2022) Elevated expression of RIT1 hyperactivates RAS/MAPK signal and sensitizes hepatocellular carcinoma to combined treatment with sorafenib and AKT inhibitor. Oncogene 41(5): 732-744.
15. Nakamura H, Aoki H, Hino O, Moriyama M (2011) HCV core protein promotes heparin binding EGF‐like growth factor expression and activates Akt. Hepatol Res 41(5): 455-462.
16. Farcas M, Gavrea AA, Gulei D, Ionescu C, Irimie A (2019) SIRT1 in the development and treatment of hepatocellular carcinoma. Front Nutr 6: 148.
17. Martins IJ (2016) Anti-aging genes improve appetite regulation and reverse cell senescence and apoptosis in global populations. Advances in Aging Research 5(1): 9-26.
18. Rifaï K, Idrissou M, Penault-Llorca F, Bignon YJ, Bernard-Gallon D (2018) Breaking down the contradictory roles of histone deacetylase SIRT1 in human breast cancer. Cancers (Basel) 10(11): 409.
19. El-Ashmawy NE, Khedr EG, Khedr NF, El-Adawy SA (2023) Emerging therapeutic strategy for mitigating cancer progression through inhibition of sirtuin-1 and epithelial-mesenchymal transition. Pathology-Research Practice 251: 154907.
20. Dai Q, Zhang C, Yuan Z, Sun Q, Jiang Y (2020) Current discovery strategies for hepatocellular carcinoma therapeutics. Expert opinion drug discovery 15(2): 243-258.
21. Shiragannavar VD, Karunakara SH, Puttahanumantharayappa LD, Sannappa Gowda NG, Santhekadur PK (2023) Unraveling Key signaling pathways altered in hepatocellular carcinoma. Gene Expr 22(1): 28-40.
22. He Y, Sun MM, Zhang GG, Yang J, Chen KS (2021) Targeting PI3K/Akt signal transduction for cancer therapy. Signal transduction targeted therapy 6(1): 425.
23. Carlomagno F, Chiariello M (2014) Growth factor transduction pathways: Paradigm of anti-neoplastic targeted therapy. J. Mol. Med 92: 723-733.
24. Greenwood W (2021) The role of NEDD9 in TGFβ mediated tumour initiating cell dynamics in the Claudin-low breast cancer subtype, Thesis, University of Cambridge, England, UK.
25. Takács T, Kudlik G, Kurilla A, Szeder B, Buday L, et al. (2020) The effects of mutant Ras proteins on the cell signalome. Cancer Metastasis Rev 39(4): 1051-1065.
26. Jain R, Watson U, Vasudevan L, Saini DK (2018) ERK activation pathways downstream of GPCRs. Int Rev Cell Mol Biol 338: 79-109.
27. Soriano O, Alcón-Pérez M, Vicente-Manzanares M, Castellano E (2021) The crossroads between RAS and RHO signaling pathways in cellular transformation, motility and contraction. Genes 12(6): 819.
28. Moon H, Ro SW (2021) MAPK/ERK signaling pathway in hepatocellular carcinoma. Cancers (Basel) 13(12): 3026.
29. Mirzaei H, Khodadad N, Karami C, Pirmoradi R, Khanizadeh S (2020) The AP‐1 pathway; A key regulator of cellular transformation modulated by oncogenic viruses. Rev Med Virol 30(1): e2088.
30. Xiao H, Wang G, Zhao M, Shuai W, Ouyang L, et al. (2023) Ras superfamily GTPase activating proteins in cancer: Potential therapeutic targets? Eur. J. Med Chem 248: 115104.
31. Seetharaman Thillai Villalan D (2020) RAS small GTPase signalling to the enigmatic RASSF death effectors, Thesis, Montreal University, Quebec, Canada.
32. Song Y, Bi Z, Liu Y, Qin F, Wei Y, et al. (2022) Targeting RAS-RAF-MEK-ERK signaling pathway in human cancer: Current status in clinical trials. Genes & Diseases 10(1): 76-88.
33. Punekar SR, Velcheti V, Neel BG, Wong KK (2022) The current state of the art and future trends in RAS-targeted cancer therapies. Nature Reviews Clinical Oncology 19(10): 637-655.
34. Charette N, Vandeputte C, Stärkel P (2014) Ras in digestive oncology: From molecular biology to clinical implications. Curr Opin Oncol 26(4): 454-461.
35. Akula SM, Abrams SL, Steelman LS, Emma MR, Augello G, et al. (2019) RAS/RAF/MEK/ERK, PI3K/PTEN/AKT/mTORC1 and TP53 pathways and regulatory miRs as therapeutic targets in hepatocellular carcinoma. Expert Opin Ther Targets 23(11): 915-929.
36. Dimri M, Satyanarayana A (2020) Molecular signaling pathways and therapeutic targets in hepatocellular carcinoma. Cancers (Basel) 12(2): 491.
37. Chen L, Feng J, Xu B, Zhou Y, Zheng X, et al. (2018) Expression of B7-H6 expression in human hepatocellular carcinoma and its clinical significance. Cancer Cell Int 18(1): 1-10.
38. Samatar AA, Poulikakos PI (2014) Targeting RAS-ERK signalling in cancer: Promises and challenges. Nature Reviews Drug Discovery 13(12): 928-942.
39. Roskoski R Jr (2018) Targeting oncogenic Raf protein-serine/threonine kinases in human cancers. Pharmacol Res 135: 239-258.
40. Muñoz-Maldonado C, Zimmer Y, Medová M (2019) A comparative analysis of individual RAS mutations in cancer biology. Front Oncol 9: 1088.
41. Yan Q, Yao G (2021) A network pharmacology-based study on the anti-hepatoma effect of Phellodendri Chinensis Cortex. Asian Journal of Traditional Medicines 16(5): 276-293.
42. Reiner DJ, Lundquist EA (2018) Small GTPases. The Online Review of elegans Biology, WormBook, Pasadena, California, USA.
43. Maertens O, Cichowski K (2014) An expanding role for RAS GTPase activating proteins (RAS GAPs) in cancer. Advances in Biological Regulation 55: 1-14.
44. Chen H, Yang Y, Wang J, Shen D, Zhao J, et al. (2018) miR-130b-5p promotes proliferation, migration and invasion of gastric cancer cells via targeting RASAL1. Oncol Lett 15(5): 6361-6367.
45. Zhang X, Li N, Li X, Zhao W, Qiao Y, et al. (2012) Low expression of DAB2IP contributes to malignant development and poor prognosis in hepatocellular carcinoma. J Gastroenterol Hepatol 27(6): 1117-1125.
46. He H, Huang J, Wu S, Jiang S, Liang L, et al. (2021) The roles of GTPase-activating proteins in regulated cell death and tumor immunity. J Hematol Oncol 14: 1-1
47. Sobhani N, Tardiel-Cyril DR, Davtyan A, Generali D, Roudi R, et al. (2021) CTLA-4 in regulatory T cells for cancer immunotherapy. Cancer 13(6): 1440.
48. Liu Z, Yu Y, Huang Z, Kong Y, Hu X, Xiao W, et al. (2019) CircRNA-5692 inhibits the progression of hepatocellular carcinoma by sponging miR-328-5p to enhance DAB2IP expression. Cell Death Dis 10(12): 900.
49. Harrell Stewart DR (2020) A novel role for RASSF1A in the regulation of RAS activation, Electronic Theses and Dissertations, University of Louisville, Kentucky, USA.
50. Oceandy D, Mohamed TM, Cartwright EJ, Neyses L (2011) Local signals with global impacts and clinical implications: Lessons from the plasma membrane calcium pump (PMCA4). Biochim Biophys Acta 1813(5): 974-978.
51. Zinatizadeh MR, Momeni SA, Zarandi PK, Chalbatani GM, Dana H, et al. (2019) The role and function of Ras-association domain family in cancer: A Review. Genes & Diseases 6(4): 378-384.
52. Zhao P, Malik S, Xing S (2021) Epigenetic mechanisms involved in HCV-induced hepatocellular carcinoma (HCC). Front Oncol 11: 677926.
53. Luo H, Wei W, Ye Z, Zheng J, Xu Rh (2021) Liquid biopsy of methylation biomarkers in cell-free DNA. Trends Mol Med 27(5): 482-500.
54. Khan FS, Ali I, Afridi UK, Ishtiaq M, Mehmood R (2017) Epigenetic mechanisms regulating the development of hepatocellular carcinoma and their promise for therapeutics. Hepatol Int 11(1): 45-53.
55. Dong Y, Liu X, Jiang B, Wei S, Xiang B, et al. (2022) A genome-wide investigation of effects of aberrant DNA methylation on the usage of alternative promoters in hepatocellular carcinoma. Front Oncol 11: 780266.
56. Ghazi T, Arumugam T, Foolchand A, Chuturgoon AA (2020) The impact of natural dietary compounds and food-borne mycotoxins on DNA methylation and cancer. Cells 9(9): 2004.
57. Al Mahi A, Ablain J (2022) RAS pathway regulation in melanoma. Dis Model Mech 15(2): dmm049229.
58. Gong J, Yan Z, Liu Q (2020) Progress in experimental research on SPRED protein family. J Int Med Res 48(8): 0300060520929170.
59. Ma XN, Liu XY, Yang YF, Xiao FJ, Li QF, et al. (2011) Regulation of human hepatocellular carcinoma cells by Spred2 and correlative studies on its mechanism. Biochem Biophys Res Commun 410(4): 803-808.
60. Hirata Y, Brems H, Suzuki M, Kanamori M, Okada M, et al. (2016) Interaction between a domain of the negative regulator of the Ras-ERK pathway, SPRED1 protein, and the GTPase-activating protein-related domain of neurofibromin is implicated in legius syndrome and neurofibromatosis type 1. J Biol Chem 291(7): 3124-3134.
61. Lorenzo C, McCormick F (2020) SPRED proteins and their roles in signal transduction, development, and malignancy. Genes Dev 34(21-22): 1410-1421.
62. King JA, Corcoran NM, Giovanna M, Straffon AF, Smith CT, et al. (2006) Eve-3: A liver enriched suppressor of Ras/MAPK signaling. J Hepatol 44(4): 758-767.
63. Markegard EC (2018) Oncogene-mediated inhibition of the Spred1 tumor suppressor, UC San Francisco Electronic Theses and Dissertations, The University of California, San Francisco, USA.
64. Motta M, Fasano G, Gredy S, Brinkmann J, Bonnard AA, et al. (2021) SPRED2 loss-of-function causes a recessive Noonan syndrome-like phenotype. The American Journal of Human Genetics 108(11): 2112-2129.
65. Francavilla C, O Brien CS (2022) Fibroblast growth factor receptor signalling dysregulation and targeting in breast cancer. Open Biology 12(2): 210373.
66. Asl ER, Amini M, Najafi S, Mansoori B, Mokhtarzadeh A, et al. (2021) Interplay between MAPK/ERK signaling pathway and MicroRNAs: A crucial mechanism regulating cancer cell metabolism and tumor progression. Life Sci 278: 119499.
67. Sun J, Yoon J, Lee M, Hwang YS, Daar IO (2020) Sprouty2 regulates positioning of retinal progenitors through suppressing the Ras/Raf/MAPK pathway. Sci Rep10(1): 1-12.
68. Kawazoe T, Taniguchi K (2019) The Sprouty/Spred family as tumor suppressors: Coming of age. Cancer Sci 110(5): 1525-1535.
69. Lee SA, Ladu S, Evert M, Dombrowski F, De Murtas V, et al. (2010) Synergistic role of sprouty2 inactivation and c‐Met up‐regulation in mouse and human hepatocarcinogenesis. Hepatology 52(2): 506-517.
70. Bivona TG (2019) Dampening oncogenic RAS signaling. Science 363(6433): 1280-1281.
71. García-Alonso S, Mesa P, de la Puente Ovejero L, Aizpurua G, Lechuga CG, et al. (2022) Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation. Mol. Cell 82(18): 3438-3452. e8.
72. Chuphal B (2022) Molecular genetics of cancer. Genetics Fundamentals Notes, Springer, Singapore, pp. 871-952.
73. Xu Y, Chen X, Pan S, Wang ZW, Zhu X (2021) TM7SF2 regulates cell proliferation and apoptosis by activation of C-Raf/ERK pathway in cervical cancer. Cell Death Discovery 7(1): 299.
74. Lin Z, Jianhua Z, Kai W, Yanhong H, Haorun L (2022) Effects of Raf kinase inhibitor protein on biological characteristics of hepatocellular carcinoma cells and its potential therapeutic effects. iLIVER 1(4): 275-282.
75. Yesilkanal AE (2019) Discovery of Anti-metastatic therapies guided by the physiological suppressor Raf kinase inhibitory protein. Dissertation, University of Chicago, Illinois, USA.
76. Cessna H, Baritaki S, Zaravinos A, Bonavida B (2022) The Role of RKIP in the Regulation of EMT in the tumor microenvironment. Cancers (Basel) 14(19): 4596.
77. Park HB, Baek KH (2022) E3 ligases and deubiquitinating enzymes regulating the MAPK signaling pathway in cancers. Biochimica Biophysica Acta (BBA)-Reviews on Cancer 1877(3): 188736.
78. Mandal JP, Shiue CN, Chen Y C, Lee MC, Yang HH, et al. (2021) PKCδ mediates mitochondrial ROS generation and oxidation of HSP60 to relieve RKIP inhibition on MAPK pathway for HCC progression. Free Radic Biol Med 163: 69-87.
79. Maik-Rachline G, Hacohen-Lev-Ran A, Seger R (2019) Nuclear ERK: Mechanism of translocation, substrates, and role in cancer. Int J Mol Sci 20(5): 1194.
80. Lin Y, Lin F, Zhang Z, Peng L, Yang W, et al. (2023) The FGFR1 signaling pathway upregulates the oncogenic transcription factor FOXQ1 to promote breast cancer cell growth. Int J Biol Sci19(3): 744-759.
81. Ascenção K, Szabo C (2022) Emerging roles of cystathionine β-synthase in various forms of cancer. Redox Biology 53: 102331.
82. Cingolani F, Liu Y, Shen Y, Wen J, Farris AB, et al. (2022) Redundant functions of ERK1 and ERK2 maintain mouse liver Homeostasis through down‐regulation of bile acid synthesis. Hepatology Communications 6(5): 980-994.
83. Emami Nejad A, Najafgholian S, Rostami A, Sistani A, Shojaeifar S, et al. (2021) The role of hypoxia in the tumor microenvironment and development of cancer stem cell: A novel approach to developing treatment. Cancer Cell Int 21(1): 62.
84. Wilczek MP, Armstrong FJ, Geohegan RP, Mayberry CL, DuShane JK, et al. (2021) The MAPK/ERK Pathway and the Role of DUSP1 in JCPyV Infection of Primary Astrocytes. Viruses 13(9): 1834.
85. Chen HF, Chuang HC, Tan TH (2019) Regulation of Dual-Specificity Phosphatase (DUSP) ubiquitination and protein stability. Int J Mol Sci 20(11): 2668.
86. Castellano E, Downward J (2010) Role of RAS in the regulation of PI 3-kinase. Phosphoinositide 3-kinase in Health and Disease, Springer, Berlin, Germany, pp. 143-169.
87. Wang B, Zheng B, Cao L, Liao K, Huang D, et al. (2021) T-lymphoma invasion and metastasis 1 promotes invadopodia formation and is regulated by the PI3K/Akt signaling pathway in hepatocellular carcinoma. Exp Cell Res 407(2): 112806.
88. Li Q, Li Z, Luo T, Shi H (2022) Targeting the PI3K/AKT/mTOR and RAF/MEK/ERK pathways for cancer therapy. Molecular Biomedicine 3(1): 47.
89. Sivasudhan E, Blake N, Lu ZL, Meng J, Rong R (2021) Dynamics of m6A RNA methylome on the hallmarks of hepatocellular carcinoma. Frontiers in Cell and Developmental Biology 9: 642443.
90. Cabral LK D, Tiribelli C, Sukowati CH (2020) Sorafenib resistance in hepatocellular carcinoma: The relevance of genetic heterogeneity. Cancers (Basel) 12(6): 1576.
91. Javaid S, Schaefer A, Goodwin CM, Nguyen VV, Massey FL, et al. (2022) Concurrent inhibition of ERK and farnesyltransferase suppresses the growth of HRAS mutant head and neck squamous cell carcinoma. Mol Cancer Ther 21(5): 762-774.
92. Juengpanich S, Topatana W, Lu C, Staiculescu D, Li S, et al. (2020) Role of cellular, molecular and tumor microenvironment in hepatocellular carcinoma: Possible targets and future directions in the regorafenib era. Int. J. Cancer 147(7): 1778-1792.
93. Yusra Z, Hassan M, Linh P, Wasim A, Keisaku S (2024) Advancements in immunotherapeutic treatments for hepatocellular carcinoma: Potential of combination therapies. International Journal of Molecular Sciences 25(13): 6830.