HLTF Promotes the Proliferation of Osteosarcoma Cells and Cisplatin Resistance

Background: Osteosarcoma, the most common primary malignant tumor of bone tissue, is characterized by aggressive biological behavior and poor clinical outcomes. The Helicase-Like Transcription Factor (HLTF), a key regulator of DNA damage response and chromatin remodeling processes, has been increasingly recognized for its crucial role in the pathogenesis and progression of various malignancies. Objective: This study aimed to elucidate the regulatory role of HLTF in modulating critical cellular processes, including proliferation, migration, and apoptosis in osteosarcoma cells, while concurrently investigating its potential as a molecular determinant of cisplatin chemoresistance. Methods: The CCK-8 and colony formation assays were carried out to systematically evaluate the impact of HLTF on the proliferative capabilities of osteosarcoma cells. Additionally, the transwell and cell scratch assays were performed to determine the effect of HLTF on the migratory potential of osteosarcoma cells. Furthermore, the CCK8 assay and the subcutaneous tumorigenesis experiment were conducted in nude mice to determine the effect of HLTF on the sensitivity of osteosarcoma cells to cisplatin. Results: Our findings revealed that silencing HLTF expression in osteosarcoma cells led to a marked suppression of both cell proliferation and invasive potential. In contrast, the overexpression of HLTF was found to augment the proliferative and migratory abilities of these cells. Remarkably, downregulating HLTF in osteosarcoma cells heightened cell sensitivity to cisplatin, which was further validated by in vivo experiments. Conclusion: Collectively, our findings strongly indicate that HLTF acts as an oncogene, actively driving the proliferation of osteosarcoma cells and conferring resistance to cisplatin. other: 4 Discussion Despite advancements in medicine that have given doctors a deeper understanding of osteosarcoma diagnosis and treatment strategies, the overall prognosis for osteosarcoma remains unsatisfactory, with mortality rates remaining high[15, 16]. The development of cancer is a complex process influenced by numerous factors. Consequently, it is crucial to investigate the underlying mechanisms of osteosarcoma to discover novel and effective targets[17]. Post-translational modification of protein plays a crucial role in the occurrence and development of tumors[6]. Research has shown that HLTF can catalyze the ubiquitination of PCNA at lys63 through the Ubc13-Mms2 E2 complex, a modification that enhances the extension of DNA polymerases and plays a crucial role in DNA replication[11, 18]. Additionally, HLTF can interact with Rad18 to promote the ubiquitination of PCNA at K164, further facilitating DNA damage repair[12]. However, the function of HLTF in tumors remains highly controversial. Previous studies have shown that HLTF suppresses tumor progression in colon cancer by DNA repair and maintaining genomic stability[19, 20]. Conversely, HLTF functions as an oncogene in renal cancer, hypopharyngeal cancer, and cervical cancer, regulating tumorigenesis and progression[21-23]. These varying functions of HLTF may be attributed to the regulation of different downstream genes and the expression of different truncated mutants in distinct tumor types. In this study, we examined the biological function of HLTF in osteosarcoma cells. We explored the association between HLTF expression and overall survival in sarcoma using the GEPIA web tool, ENCORI Pan-Cancer analysis platform, and UALCAN-TCGA database. Interestingly, all three platforms consistently demonstrated that patients with high HLTF expression in sarcoma exhibited shorter overall survival. Previous studies have shown significant elevations of HLTF in hypopharyngeal cancer and renal cancer. However, in this study, analysis using the UALCAN-TCGA database revealed no significant difference in HLTF expression between sarcoma and adjacent normal tissues. We speculate that this may be due to the limited number of normal tissue samples (n = 2) in the UALCAN-TCGA database, as well as the lack of clinical specimens to measure HLTF expression in osteosarcoma and adjacent normal tissues, which may have led to a lack of scientific validity in the results. Future studies will aim to improve the measurement of HLTF expression in clinical osteosarcoma and adjacent normal tissues. These findings suggest that HLTF may function as an oncogene involved in the regulation of sarcoma progression, but its specific biological function in sarcoma requires further investigation. To further explore the role of HLTF in osteosarcoma, we discovered that knocking down HLTF in osteosarcoma cells resulted in a significant decrease in cell proliferation and invasion ability, while overexpressing HLTF yielded opposite results. Based on these findings, we concluded that HLTF primarily functions as an oncogene in osteosarcoma. Studies have shown that HLTF inhibits tumor migration and invasion in colon cancer by regulating the TGF-β/Smad pathway[24]. Additionally, HLTF suppresses cancer cell proliferation by inhibiting DNA synthesis[25]. However, HLTF can serve as an early tumor marker in renal cancer and promote tumor cell proliferation[21]. Furthermore, high mRNA expression of HLTF in cervical cancer cells suggests its involvement in tumor transformation[26]. Therefore, the mechanism by which HLTF promotes osteosarcoma cell proliferation in this study may be similar to that in renal cancer or may involve early tumor transformation, but the specific mechanism remains unclear. Thus, further research is needed to confirm these hypotheses. Currently, the primary clinical treatment methods for osteosarcoma typically encompass personalized neoadjuvant chemotherapy, surgical treatment, and postoperative high-dose chemotherapy[16]. Cisplatin stands as one of the main DNA-damaging agents in the treatment of osteosarcoma[27]. The mechanism of action involves causing genomic DNA damage by crosslinking with nuclear DNA and inducing tumor cell apoptosis through the generation of reactive oxygen species, although most patients eventually develop resistance[28]. HLTF is a highly conserved gene that shares numerous features with S. cerevisiae RAD5, including PCNA polyubiquitination activity, ATP-dependent DNA translocase enzymatic activity, structural interactions with Rad18 homolog genes, and an ancient conserved sequence domain, the HIRAN domain, which is crucial for binding to the 3' end of ssDNA[13]. Bryant et al. have shown that Rad5 regulates tumor cell sensitivity to cisplatin[13]. Given the widespread use of platinum-based chemotherapy drugs in cancer treatment, it is imperative to further investigate the impact of HLTF dysregulation on DNA damage and platinum drug resistance. Therefore, our study examined the effect of knocking down HLTF on cisplatin sensitivity in osteosarcoma cells by in vivo and in vitro experiments. Our study showed that knocking down HLTF in osteosarcoma cells significantly increased cell sensitivity to cisplatin.