Mechanisms underlying hepatocellular carcinoma progression through N6-methyladenosine modifications of long non-coding RNA

Abstract

Hepatocellular carcinoma (HCC) is a highly lethal malignancy with limited treatment options, particularly for patients with advanced stages of the disease. Sorafenib, the standard first-line therapy, faces significant challenges due to the development of drug resistance. Yu et al explored the mechanisms by which lncRNA KIF9-AS1 regulates the stemness and sorafenib resistance in HCC using a combination of cell culture, transfection, RNA immunoprecipitation, co-immunoprecipitation, and xenograft tumor models. They demonstrate that N6-methyladenosine-modified long non-coding RNA KIF9-AS1 acts as an oncogene in HCC. This modification involves methyltransferase-like 3 and insulin-like growth factor 2 mRNA-binding protein 1, which play critical roles in regulating KIF9-AS1. Furthermore, KIF9-AS1 stabilizes and upregulates short stature homeobox 2 by promoting its deubiquitination through ubiquitin-specific peptidase 1, thereby enhancing stemness and contributing to sorafenib resistance in HCC cells. These findings provide a theoretical basis for KIF9-AS1 as a diagnostic marker and therapeutic target for HCC, highlighting the need for further investigation into its clinical application potential.

Key Words: Hepatocellular carcinoma; Stemness; Sorafenib resistance; Long non-coding RNA KIF9-AS1; Short stature homeobox 2; N6-methyladenosine

Core Tip: Yu et al present evidence showing that m6A-modified long non-coding RNA KIF9-AS1 drives the progression of hepatocellular carcinoma (HCC) and reveal novel molecular mechanisms underlying this process. The m6A modification, mediated by modification involves methyltransferase-like 3 and insulin-like growth factor insulin-like growth factor 2 mRNA-binding protein 1, stabilizes and upregulates KIF9-AS1 expression. In turn, KIF9-AS1 enhances the stability and expression of SHOX2 by promoting its deubiquitination via ubiquitin-specific peptidase 1, which strengthens stemness and contributes to sorafenib resistance in HCC cells. Future studies should further validate KIF9-AS1 as a potential diagnostic biomarker for HCC and explore its therapeutic applications in HCC treatment.

TO THE EDITOR

Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer, characterized by high global incidence and mortality rates. Several factors contribute to the development of HCC, including chronic infections with hepatitis viruses (such as hepatitis B and C), cirrhosis, alcohol abuse, and metabolic syndrome[1]. The prognosis for HCC is generally poor, with a low five-year survival rate. This is primarily due to the fact that HCC is often diagnosed at advanced stages, which delays treatment initiation[1,2]. A major challenge in HCC treatment is drug resistance, which refers to the ability of tumor cells to withstand chemotherapy, leading to chemotherapy failure and disease recurrence[3,4]. Research indicates that HCC cells evade drug effects through various mechanisms, such as inhibiting apoptosis, activating autophagy, promoting drug efflux, and undergoing epigenetic alterations[3,5]. Moreover, cancer stem cells are believed to play a pivotal role in the initiation, invasion, metastasis, and recurrence of HCC, with their presence closely linked to drug resistance[6,7]. Thus, effective therapeutic strategies for HCC must address the biological characteristics of the tumor, including stemness and drug resistance mechanisms, to enhance treatment efficacy and improve patient survival rates[8].

Non-coding RNAs, especially long non-coding RNAs (lncRNAs), play a critical role in the development of HCC. In 2014, Chen et al[9] conducted the first systematic review on the role of lncRNAs in liver cancer, elucidating their involvement in tumorigenesis via epigenetic regulation, transcriptional, and post-transcriptional modifications. Studies have demonstrated that lncRNAs influence key biological processes, such as proliferation, migration, and autophagy, by modulating various signaling pathways and gene expression[10]. Additionally, aberrant expression of lncRNAs (e.g., HULC, MALAT1) is strongly linked to the onset and progression of HCC, making them potential novel biomarkers and therapeutic targets[9,11,12]. LncRNAs not only sustain stemness, thereby promoting tumorigenesis and progression[13], but alterations in the expression of specific lncRNAs can also enhance HCC's resistance to chemotherapy drugs[8,14]. Yao et al[15] demonstrated that LINC01189 enhances the chemoresistance of HCC cells by being regulated by hsa-miR-155-5p. During the same period, the interaction between the TGF-β/EMT pathway and lncRNAs was elucidated, with lncRNAs enhancing resistance to cisplatin and sorafenib via autophagy induction[16]. In 2022, Zhang et al[17] reported that LINC01132 enhances immunosuppression and drug resistance through the NRF1/DPP4 axis, and is significantly associated with the prognosis of HCC patients. Additionally, Wnt/β-catenin pathway-related lncRNAs, such as SNHG14 and FAM83H-AS1, have been shown to promote HCC progression by regulating macrophage polarization and the tumor microenvironment[18]. These findings indicate that the modified lncRNAs significantly contribute to regulating stemness and drug tolerance in HCC cells[19]. Gaining a deeper understanding of the role of lncRNAs in HCC will not only advance our knowledge of the molecular mechanisms underlying this disease but may also provide valuable insights for developing new therapeutic strategies.

Yu et al[20] offer valuable insights into HCC, with a particular focus on the role of m6A-modified lncRNA KIF9-AS1 in regulating stemness and sorafenib resistance. Sorafenib, a first-line therapeutic for HCC, faces significant challenges due to the development of drug resistance[21]. In their study, Yu et al[20] revealed the oncogenic role of KIF9-AS1 in HCC. Specifically, they found that KIF9-AS1 expression was upregulated in HCC tissues compared to normal liver tissues. Additionally, knocking down KIF9-AS1 inhibited HCC cell stemness and reduced sorafenib resistance. These findings suggest that KIF9-AS1 could serve as both a biomarker and a therapeutic target. However, further comprehensive evaluation and validation of KIF9-AS1-targeted therapies are required.

HOW KIF9-AS1 ENHANCES STEMNESS AND RESISTANCE TO SORAFENIB

Yu et al[20] demonstrated that the modification involves methyltransferase-like 3 (METTL3) and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1)-mediated m6A modification stabilizes and enhances the expression of KIF9-AS1, highlighting the critical role of METTL3, IGF2BP1, and m6A modification in HCC treatment. m6A, one of the most prevalent RNA modifications, regulates various biological processes, including gene expression, RNA stability, and translation[22]. METTL3, as an m6A methyltransferase, adds m6A modifications to RNA, while IGF2BP1, an m6A reader protein, recognizes and binds to these modifications, influencing the stability and translation efficiency of target RNAs[23]. Numerous studies have shown that METTL3/IGF2BP1-mediated m6A modification promotes tumor cell proliferation, migration, and transformation[24,25]. However, it is important to acknowledge that other epigenetic mechanisms, such as DNA methylation and histone modifications, may also contribute to HCC drug resistance. Further research is needed to fully elucidate these epigenetic mechanisms and their impact on HCC drug resistance.

Yu et al[20] also explored the downstream regulatory mechanisms of KIF9-AS1 in HCC. They discovered that KIF9-AS1 enhances the stability and expression of short stature homeobox 2 (SHOX2) by promoting deubiquitination via ubiquitin-specific peptidase 1 (USP1). SHOX2, an oncogene implicated in tumor progression and metastasis, has been linked to several cancers, including lung, prostate, and breast cancer[26-28]. Furthermore, SHOX2 has been shown to contribute to drug resistance in lung cancer[26]. Elevated SHOX2 expression has been observed in HCC patients and is associated with tumor recurrence[29]. High expression of SHOX2 enhances the stemness phenotype of liver cancer cells, with the marker CD13 being highly expressed, endowing cells with proliferative, migratory, and adhesive capabilities. Further molecular mechanism studies have revealed that CD13 can interact with histone deacetylase 5 (HDAC5), inhibiting its ubiquitination and degradation, thereby enhancing the protein stability of HDAC5. This process leads to HDAC5-mediated deacetylation of lysine-specific demethylase LSD1, enhancing LSD1's activity[30]. LSD1, in turn, stabilizes the NF-κB subunit p65 protein and promotes its nuclear translocation by reducing the methylation level of p65, ultimately activating the transcription of downstream oncogenic genes of NF-κB[31]. This cascade reaction results in decreased sensitivity of tumor cells to sorafenib. Studies have shown that the combination of the CD13 inhibitor ubenimex with sorafenib can significantly inhibit tumor growth and reverse drug resistance[30]. Therefore, SHOX2 and CD13 can serve as a biomarker for predicting the prognosis of postoperative HCC patients and their responsiveness to sorafenib therapy.

USP1, a deubiquitinating enzyme, is abnormally expressed in various cancers, particularly in HCC, where its expression is significantly higher than in normal tissues. High USP1 expression correlates with increased tumor aggressiveness, metastasis, and reduced patient survival[32]. As a result, USP1 is considered a potential therapeutic target in HCC, and its value as a prognostic biomarker is widely recognized. Although SHOX2 is known to be a substrate for USP1-mediated deubiquitination, it remains unclear whether KIF9-AS1 regulates SHOX2 expression through USP1, or if other epigenetic modifications play a more prominent role. Further investigation is needed to clarify this relationship.

CLINICAL IMPLICATIONS

Yu et al[20] found that KIF9-AS1 upregulates the expression of SHOX2 through m6A modification, thus aiding in the specific diagnosis of early-stage HCC by detecting the epigenetic modification levels of m6A-related molecules KIF9-AS1 and SHOX2 in tumor tissues or blood. Furthermore, upregulated expression of KIF9-AS1 in clinical samples is significantly associated with poor overall survival in HCC patients. Moreover, high KIF9-AS1 expression in patients with advanced HCC strongly correlates with tumor-lymph node metastasis. Therefore, KIF9-AS1 can also serve as a biomarker for assessing the prognosis of HCC patients. As previously mentioned, KIF9-AS1 promotes the stemness characteristics of HCC cells by upregulating SHOX2, thereby enhancing resistance to sorafenib[30]. Detecting the levels of KIF9-AS1 and its downstream targets SHOX2 and CD31 in HCC patients will aid in predicting their response to sorafenib treatment. Correspondingly, adopting therapeutic measures targeting these markers is expected to optimize HCC treatment strategies and improve patient outcomes. Studies have shown that METTL3 inhibitors exert potent anti-cancer effects by reducing m6A modification function, potentially reducing drug resistance[33]. These findings suggest that KIF9-AS1 plays a key role in HCC progression, reinforcing its potential as both a biomarker and a therapeutic target. Currently, some m6A-related drugs have entered early-stage clinical trials, but the clinical translation of lncRNA applications still faces challenges such as specific recognition, optimization of delivery systems, and validation through large-scale clinical trials. Meanwhile, no significant association was observed between KIF9-AS1 expression and factors such as gender, age, or tumor size. In light of this, more direct experimental validation is needed to confirm its therapeutic potential, thereby promoting the development of precision medicine.

CONCLUSION

The study by Yu et al[20] not only elucidates the role of KIF9-AS1 in HCC but also provides a theoretical framework for developing new therapeutic strategies. As an m6A-modified lncRNA, KIF9-AS1 promotes stemness and sorafenib resistance in HCC by facilitating USP1-mediated deubiquitination of SHOX2. This finding highlights new potential targets for clinical treatment, which could improve the prognosis for HCC patients. However, Yu et al[20] did not fully elucidate the specific signaling pathways downstream of SHOX2, nor did they explore therapeutic strategies targeting the KIF9-AS1/SHOX2 axis or its therapeutic potential. Future research should focus on deepening the mechanistic investigation of the downstream pathways of SHOX2 and their causal relationship with HCC stemness. Utilizing single-cell sequencing or spatial transcriptomics technology could help dissect the cell-specific role of the KIF9-AS1/SHOX2 axis within the tumor microenvironment. Additionally, developing targeted intervention strategies, such as using m6A inhibitors (e.g., targeting METTL3) or SHOX2 antagonists, and testing their efficacy in patient-derived or organoid models, could provide valuable insights.