Mina Baek, Minjae Kim, Hae In Choi, Bert Binas, June Cha, Kyoung Hwa Jung, Sungkyoung Choi, Young Gyu Chai
Abstract
The multikinase inhibitor sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but many patients become sorafenib-resistant (SR). This study investigated the efficacy of another kinase inhibitor, regorafenib (Rego), as a second-line treatment. We produced SR HCC cells, wherein the PI3K-Akt, TNF, cAMP, and TGF-beta signaling pathways were affected. Acute Rego treatment of these cells reversed the expression of genes involved in TGF-beta signaling but further increased the expression of genes involved in PI3K-Akt signaling. Additionally, Rego reversed the expression of genes involved in nucleosome assembly and epigenetic gene expression. Weighted gene co-expression network analysis (WGCNA) revealed four differentially expressed long non-coding RNA (DElncRNA) modules that were associated with the effectiveness of Rego on SR cells. Eleven putative DElncRNAs with distinct expression patterns were identified. We associated each module with DEmRNAs of the same pattern, thus obtaining DElncRNA/DEmRNA co-expression modules. We discuss the potential significance of each module. These findings provide insights and resources for further investigation into the potential mechanisms underlying the response of SR HCC cells to Rego.
Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver cancer and closely associated with chronic liver disease and cirrhosis [1]. An important first-line treatment for HCC is sorafenib [2], a multi-targeted tyrosine kinase inhibitor that suppresses cell proliferation and angiogenesis in advanced HCC. However, most patients become sorafenib-resistant (SR) within 6 months of treatment [3], leading to the need for second-line treatment options such as regorafenib (Rego) [4, 5]. However, the molecular mechanisms of SR and Rego remain poorly understood, hindering the further improvement of treatment options. Long non-coding RNAs (lncRNAs) are a class of RNA molecules that are over 200 nucleotides in length and do not encode proteins [6, 7]. They play critical roles in the development and progression of various cancer types by regulating gene expression, signaling pathways, and epigenetic modifications [8]. LncRNAs have also been identified as potential targets to overcome drug resistance in cancer cells [9], and recent research demonstrated that targeting specific lncRNAs can enhance the efficacy of cancer treatment by sensitizing drug-resistant cancer cells [10]. The role of lncRNAs has also been explored specifically in SR HCC cells. For instance, MALAT1 [11] and NEAT1 [12] were found to be upregulated in HCC cells with acquired SR, and their knockdown resulted in re-sensitization to sorafenib. Similarly, SNHG1 was upregulated in SR HCC cells, and its knockdown improved sorafenib sensitivity by promoting apoptosis and suppressing cell migration [13]. These studies demonstrate the involvement of lncRNAs in the development of drug resistance in cancer cells, including SR HCC cells, and suggest that targeting specific lncRNAs may be a promising strategy to overcome SR.
Materials and methods
Cell culture and treatments
The HCC cell line Huh7 was purchased from the Korean Cell Line Bank (Seoul, Korea) and cultured in MEM supplemented with 10% heat-inactivated FBS, penicillin, and streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in a humidified atmosphere with 95% air and 5% CO2. (Thermo Scientific, HERAcell 240, MA, USA) To establish SR, the cells were cultured for ~6 months in the presence of sorafenib, starting at 1 μM and with increments of 0.25 μM per passage until 5 μM. In order to assess acute drug effects on regular Huh7 cells (Con) or SR Huh7 cells, the cells were freshly seeded into the respective conditions as described in the subsections further below.
Results
Establishment and verification of SR HCC cells
The study was initiated by the establishment of a sorafenib-resistant (SR) HCC line. In this regard, Huh7 cells were chronically exposed to increasing concentrations of sorafenib, reaching a plateau at 5 μM (see Materials and Methods). Subsequently, drug resistance was quantified through viability and plating assays over 48 hours and ≥ 1 week, respectively. As expected, viability of the SR cells was compromised only when the sorafenib concentration exceeded 5 μM, while the original Huh7 cells showed a significant death rate already at 1 μM (Fig 1A). Likewise, colony formation by the SR cells was not compromised at up to 5 μM, but was largely lost at that concentration in the control cells (Fig 1B). On the other hand, compared to the untreated (DMSO) control cells, the SR cells exhibited a reduced plating efficiency (Fig 1B) and a smaller S-phase/larger G1-phase fraction regardless of the acute absence or presence of (up to 5 μM) sorafenib (Fig 1C). A similar S-phase reduction was caused by the acute treatment of the control cells, while no further reduction was seen in the SR cells (Fig 1C).
Discussion
In this study, we aimed to understand how regorafenib treatment affects the gene expression profiles of HCCs in the context of SR. By examining DEmRNAs and DElncRNAs, we gained insights into the molecular changes and potential mechanisms underlying the response to regorafenib in SR cells. To accomplish this objective, we generated SR cells and confirmed their reduced sensitivity and reduced responsiveness to sorafenib through cell viability, apoptosis, and colony formation assays. It is known that the SR of HCC involves various signaling pathways [23], amongst them the activation of the PI3K/Akt pathway, which facilitates their survival, impedes apoptosis, and boosts growth [24, 25]. The present study confirms the upregulation of PI3K-Akt signaling in SR cells (S4 Fig and Fig 3). Typically, upregulation of PI3K-Akt signaling is associated with increased cell invasion or migration [26]. However, our GO results did not support such a relationship. This reveals an important limitation of regorafenib that needs to be addressed in the future but also indicates that its anti-tumor activity in the context of SR involves alternative mechanisms.
Conclusions
Using a cell culture model, we characterized the acute transcriptional response of SR HCC cells to the second-line chemotherapeutic, regorafenib. In this context, we identified regorafenib-responsive signaling pathways and DElncRNAs/DEmRNAs co-expression modules with regulatory potential. From a clinical perspective, some effects appear beneficial but others are reason for concern. Future work will need to study the long-term exposure of SR HCC cells to regorafenib.
Citation: Baek M, Kim M, Choi HI, Binas B, Cha J, Jung KH, et al. (2024) Identification of differentially expressed mRNA/lncRNA modules in acutely regorafenib-treated sorafenib-resistant Huh7 hepatocellular carcinoma cells. PLoS ONE 19(4): e0301663. https://doi.org/10.1371/journal.pone.0301663
Editor: Bashdar Mahmoud Hussain, Hawler Medical University, IRAQ
Received: December 3, 2023; Accepted: March 20, 2024; Published: April 11, 2024
Copyright: © 2024 Baek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The RNA-seq data used and analyzed during the current study are included in this published article and its Additional files. The open RNA-seq data (Accession number: GSE242333) is available in the NCBI database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE242333).
Funding: This work was supported by the National Research Foundation of Korea (NRF) Grants 2017M3A9G7073033 and 2020R1A2C1014193 (to Y. G. C.), and 2020R1F1A1063217 (to B. B.) from the Korean government. This work was supported by Institute of Information & communications Technology Planning & Evaluation (IITP) grant funded by the Korea government (MSIT) (No.RS-2022-00155885, Artificial Intelligence Convergence Innovation Human Resources Development (Hanyang University ERICA)).]. Role of Funder statement Young Gyu Chai – Conceptualization and Supervision Bert Binas – Data curation and Writing Sungkyoung Choi – Methodology and Software.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: AKT, Protein kinase B signaling; BP, Biological process; CC, Cellular Component; DAVID, Database for Annotation, Visualization, and Integrated Discovery; DElncRNAs, Differentially expressed lncRNAs; DEmRNAs, Differentially expressed mRNAs; GO, Gene Ontology; HCC, Hepatocellular carcinoma; IPA, Ingenuity Pathway Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; KOBAS, KEGG Orthology Based Annotation System; LncRNA, Long non-coding RNA; MF, Molecular Functions; qRT-PCR, Quantitative reverse transcription PCR; Rego, Regorafenib; RNA-seq, RNA sequencing; SR, Sorafenib resistant; SR+Rego, Rego-treated SR; WGCNA, Weighted gene co-expression network analysis
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0301663#abstract0
