Transcriptomic analysis associated with reversal of cisplatin sensitivity in drug resistant osteosarcoma cells after a drug holiday
Resistance to chemotherapy is one of the major hurdles in current cancer therapy. With the increasing occurrence of drug resistance, a paradigm shift in treatment strategy is required. Recently “medication vacation” has emerged as a unique, yet uncomplicated strategy in which withdrawal of drug pressure for certain duration allowed tumor cells to regain sensitivity to the drug. However, little is known about the molecular alterations associated with such an outcome.
In this study, human osteosarcoma (OS) cells resistant to the extensively used drug cisplatin, were withdrawn from drug pressure, and thereafter cytotoxic response of the cells to the drug was evaluated. We further performed next-generation RNA sequencing and compared transcriptome between parental (OS), resistant (OS-R) and the drug withdrawn (OS-DW) cells. Differentially expressed transcripts were identified, and biological association network (BAN), gene ontology (GO) and pathway enrichment analysis of the differentially regulated transcripts were performed to identify key events associated with withdrawal of drug pressure.
Following drug withdrawal, the sensitivity of the cells to the drug was found to be regained. Analysis of the expression profile showed that key genes like, IRAK3, IL6ST, RELA, AKT1, FKBP1A and ADIPOQ went significantly down in OS-DW cells when compared to OS-R. Also, genes involved in Wnt signaling, PI3K-Akt, Notch signaling, and ABC transporters were drastically down-regulated in OS-DW cells compared to OS-R. Although, a very small subset of genes maintained similar expression pattern between OS, OS-R and OS-DW, nonetheless majority of the transcriptomic pattern of OS-DW was distinctively different and unique in comparison to either the drug sensitive OS or drug resistant OS-R cells.
Our data suggests that though drug withdrawal causes reversal of sensitivity, the transcriptomic pattern does not necessarily show significant match with resistant or parental control cells. We strongly believe that exploration of the molecular basis of drug holiday might facilitate additional potential alternative treatment options for aggressive and resistant cancers.
KeywordsOsteosarcoma RNA-sequencing Drug-resistance Drug-holiday
Biological Association Network
Gene Expression Omnibus
Osteosarcoma drug withdrawn
Osteosarcoma extended persisters
Drug resistance remains to be one of the major challenge impeding the success of anticancer therapy in achieving prolonged survival. Multiple mechanisms of drug resistance co-exist within a single patient or even a tumour escaping chemotherapy [1, 2]. According to the classic Goldie–Coldman hypothesis of drug resistance, mutations can be spontaneously acquired by a tumour over time leading to accumulation of drug-resistant clones. Further, resistant variants in a heterogeneous tumor can be selected in a Darwinian process, or a subpopulation of the intrinsically resistant cell might cause re-growth of the tumor [3, 4]. Despite diverse mode of action, cancer cells learn to thrive on the drug treatment itself, shrugging off drug scare and continue to grow. Hence, in spite of medical advancements, a vast majority of chemotherapies inevitably fail. Identifying alternative routes by which we can overcome resistance is therefore critical. Interestingly, various pre-clinical studies have reported that cancer cell resistance to a drug is not necessarily linked with genetic mutations associated with drug targets; sometimes the resistant cancers may derive their naturally selected “fitness” through regulation of expression of transcripts post-exposure to drugs that were meant to reduce their fitness [5, 6, 7, 8, 9]. Therefore, it is proposed that if these cells are given a ‘drug break’ they eventually might revoke their sensitivity to the drug.
Drug re-challenge has been a well acknowledged old concept. Early studies were reported on drug re-challenge in small-cell lung cancers, various leukaemia’s and also following adjuvant treatment in breast cancer [10, 11, 12]. Drug re-challenge has also been in vogue not just in cancers, but also in diseases like a polycystic liver disease where the cessation of treatment led to a rebound in effect . In recent times, similar re-challenge-like routines has been used for the treatment of metastatic breast cancer and platinum-based therapy in ovarian cancers [14, 15]. As conventional therapies are still not completely effective in suppression of tumour growth, and recurrence is a common phenomenon, it is therefore imperative to explore the molecular drivers of the re-sensitization process. Till date, molecular changes associated with drug holiday hasn’t been investigated thoroughly in spite of its potential therapeutic implications. Also, if at all there are forces that allow the sensitivity to reverse, what are the possible molecular events that mark it still remains unknown. Further, the understanding of how to design intermittent therapies, what should be the ideal duration of it for sensitization of resistant cancer types, is least understood.
In our initial study, we characterized the progressive variation in transcriptomic expression pattern of osteosarcoma (OS) cells as it progressed towards acquisition of resistance to cisplatin . OS cells were selected for the study as drug resistance still remains a major obstacle to successful treatment in OS [17, 18, 19]. Post RNA sequencing, transcriptomic expression pattern was analyzed and compared between the following samples- untreated tumor cells (OS); non-dividing persisters representing the tolerant cells surviving drug shock (OS-P); extended persisters (OS-EP) denoting the proliferative cells that have revived from drug shock; and the drug resistant cells (OS-R), derived from the OS-EPs, after repeated cycles of exposure to the drug followed by clonal selection of surviving cells. Importantly, the IC50 of cisplatin in OS cells was found to be 35 μM, however at similar concentrations only ~ 20% cell death was observed in OS-R cells; the resistant cells thus showed decreased sensitivity to cisplatin at IC50 dose compared to parental OS cells . De-regulation of a key network of genes, involved in the regulation of several pathways, was observed in the OS-R cells . As a followup to our initial analysis, in the current study, we wanted to explore whether a drug break reverts the sensitivity in OS-R cells, and if so, what is the molecular basis of such regulation. The resistant cells were cultured under continuous drug pressure; therefore, in this study the OS-R cells were given a drug break and then exposed to cisplatin again to assess sensitivity. Thereafter, next-generation RNA sequencing was performed followed by comparative transcriptomic expression analysis between the drug withdrawn cells (OS-DW), OS and OS-R cells. We provide key insights into molecular events associated with reversal of cisplatin sensitivity in OS cells. This may help in re-orientation of drug treatment strategy against resistant tumor types.
Cell culture and generation of drug resistant cell line
The human osteosarcoma cell line (HOS-CRL-1543) was procured from NCCS, Pune, India, and cultured at 37 °C, 5% CO2, in minimal essential medium (HiMedia) supplemented with 10% fetal bovine serum (Invitrogen). The cell line identity was authenticated through STR profiling at Lifecode Technologies Private Limited, New Delhi, India (Project ID: M-1066). The cells were periodically monitored for any contamination. The detailed methodology for the generation of cisplatin resistant OS cells (OS-R) from HOS-CRL-1543 is described in our earlier study . In brief, the parental OS cells were exposed to an acute dose of cisplatin (1 mg/ml) which resulted in survival of a small population of cells; these drug tolerant cells were labeled as “persisters” (OS-P). The persister cells were slow dividing, but over time they revived their population to reach 90% confluency; these cells were termed as the “extended persisters (OS-EP). This cycle of drug exposure followed by revival was repeated to subsequently generate the cisplatin resistant (OS-R) cells .
Establishment of sensitivity in OS-R cells through a drug holiday
The OS-R cells were given a drug break to induce reversal of sensitivity to the drug. These cells were cultured in the absence of the drug cisplatin for seven days. The cell viability assay was thereafter performed to analyze sensitivity to cisplatin.
Cell viability assay
In vitro analysis of cytotoxicity to cisplatin was evaluated through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5di-phenyltetrazolium bromide) assay following procedures described earlier in Chowdhury et al 2009 . Briefly, cells were cultured overnight and thereafter treated with cisplatin for 24 h. Cells were incubated with MTT followed by DMSO solubilization of formazan crystals formed by the live cells. Readings were taken at 495 nm with a differential filter of 630 nm.
RNA isolation and quantitative PCR
Total RNA from the cell extracts was obtained through TRIzol reagent (Invitrogen). Complementary DNA (cDNA) synthesis was carried out using GeneSure First Strand cDNA Synthesis kit (Genetix) using oligodT. cDNA for specific genes was amplified and detected using SYBR Green (Bio-Rad) in real time PCR System (Bio-Rad). Livak method was used to quantitate the relative RNA expression level .
mRNA sequencing and analysis
RNA sequencing was conducted at Bionivid Technology Pvt. Ltd., Bangalore using Illumina HiSEQ2500 platform. The raw data was deposited to NCBI’s Gene Expression Omnibus (GEO) with GEO Series accession number GSE86053 . The detailed methodology followed for sequencing and analysis is described in Niveditha et al 2019 . Briefly, cDNA library was prepared and deep sequencing was performed to generate reads which were thereafter aligned to the human genome using TopHat (v2.0.11). Cufflinks was used to obtain expression statistics of each transcript and CuffDiff was used to collect differential gene expression data. Fold change (FC) of 1.5 and above, and a p-value cutoff of ≤0.05 was considered for differentially expressed transcripts, whereas, transcripts with log 2-fold change greater than or less than 10 were considered as significantly up or down-regulated respectively. Clustering of transcripts showing differential expression was performed on DAVID server; Cytoscape V 2.8 was applied to visualize the network, and finally, functional annotations were carried out based on Gene Ontology (GO). The differentially expressed transcripts were segregated into three functional domains- receptor-mediated signaling, intracellular signaling, and regulation of intra-cellular processes based on their biological functions, as per GO database. The transcripts showing association with all the three functional domains were considered as “key genes” .
Resistant OS-R cells regain sensitivity to cisplatin after drug withdrawal
We obtained OS-R cells as described in our previously published study . The OS-R cells were cultured in drug withdrawn media following procedure as described in materials and methods. The sensitivity of the cells (now labelled as OS-DW) to cisplatin, 24 h post treatment was thereafter evaluated. The IC50 of OS cells to cisplatin was earlier reported to be ~ 35 μM, at a similar dose, only around 20% cell death was observed in OS-R cells; the IC50 of OS-R cells was observed to be ~ 80 μM [16, 23]. However, post culture of OS-R cells in drug withdrawn media the OS-DW cells regained sensitivity to cisplatin. The IC50 of cisplatin in OS-DW cells was found to be around 37 μM which was very close to the IC50 of cisplatin observed in parental untreated OS cells. We were therefore interested to understand whether the transcriptome of OS-DW cells revert to a pre-treatment state after withdrawal, or there is a unique third state representing the cells after drug holiday. Transcriptomic analysis was therefore performed in OS, OS-R and OS-DW cells and the transcriptomic pattern was compared to understand alterations associated with re-gained sensitivity.
Comparative transcriptomic analysis between OS-DW with OS-R cells
The biological significance of differentially expressed transcripts in OS-DW cells compared to resistant OS-R cells
Comparative transcriptomic analysis between parental OS cells and OS-DW cells
The biological significance of differentially expressed transcripts in OS-DW cells compared to parental OS cells
It is widely accepted that random genetic alterations confer resistance to drugs. However, this fails to explain an increasingly observed phenomenon associated with chemotherapy against cancers- “re-treatment or re-challenge response”. It is proposed that the cells that survive a cytotoxic drug shock, when given a “drug break,” eventually salvage their sensitivity to the drug. The above observation indicates that not necessarily, resistance to drugs is a resultant of stable genetic alterations but may include a reversible state. The potential benefit of treatments with a “drug break” has been addressed in two large phase III clinical trials for docetaxel drug, in androgen-independent prostate cancer patients, where intermittent chemotherapy associated with a drug holiday reduced the level of drug toxicity and improved the quality of life [50, 51, 52]. Similar results were also obtained with metastatic renal carcinoma, treatment-resistant melanoma and other cancers, where a drug holiday led to a secondary response. This implies that in patients with a drug break, the resistant tumor cells might shed their resistance property [53, 54]. Therefore, identification of key cellular processes triggered by the withdrawal of drug can be exploited with existing drugs to trigger maximal cell death.
Earlier, Sharma et al. in 2010 performed a similar study where drug-sensitive cells were treated with drugs at concentrations exceeding the IC50 values . Following multiple rounds of treatments, they detected “drug-tolerant” cells demonstrating reduced drug sensitivity. However, further analyses revealed that the tolerant state was not stable. The tolerant cells-maintained survival by insulin-like growth factor 1 receptor signalling, in an altered chromatin state. In corroboration to above, our study also provides compelling evidence that in OS cells as well, the drug-resistant state is not permanent but is reversible, and the withdrawal of drug pressure is sufficient to induce a transcriptomic make-over effectively re-sensitizing the cells. Hence, this type of “stop and go” strategy provides an appealing treatment option for resistant OS cells. The findings not only have clinical significance, as a drug break may provide respite to the patients from persistent toxic side effects of the drug, but it also has significant economic implications as well, as patients on the intermittent therapy might spend much lesser amount on the anti-cancer drugs, which are overtly expensive, yet achieving clinical remission. However, for some medications, like targeted therapies with drugs for EGFR+ or tumors having multiple metastases before the beginning of first line treatment, continuous therapy might be more favorable than structured treatment interruptions. Therefore, the strategy must depend on the tumor type, stage of the tumor, choice of drugs and also on the patient response. Here in, for the first time we report that resistant osteosarcoma can be re-sensitized by treatment vacations from DNA damaging drugs, like, cisplatin.
Withdrawal of drug pressure in OS cells facilitates their re-sensitization to the drug- cisplatin. The transcriptomic pattern of the drug withdrawn OS-DW cells is unique and do not have striking similarity with either parental OS or resistant OS cells.
We acknowledge DST-SERB and DBT grant of RC for providing financial support for the study. We further thank Bionivid, India for performing RNA sequencing of the samples. We thank BITS-Pilani, Pilani campus for providing infrastructural support.
DN carried out the majority of data analysis including network and regulatory studies and drafted the manuscript. HS carried out differential expression analysis. SM (Syamantak) and SM (Sudeshna) participated in the design of the wet lab study and helped to draft the manuscript. RC structured the design of the study and coordination and helped to draft the manuscript. SC helped to coordinate the bioinformatics analysis and drafting of the manuscript. All authors have read and approved the final manuscript.
We thank DST-SERB (Sanction order No. EMR/2016/006788) and DBT grant (BT/PR/8799/MED/30/1067/2013) of RC for providing financial support for the study. The funding bodies had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.
Ethics approval and consent to participate
The Ethics approval from Birla Institute of Technology and Science, Pilani is not required to use human cell lines used in this study.
Consent for publication
The authors declare that they have no competing interests.
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