Heterozygous retinoblastoma gene mutation compromises in vitro osteogenesis of adipose mesenchymal stem cells – a temporal gene expression study

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Highlights

  • RB1 gene is important for osteogenic differentiation of mesenchymal stem cells.

  • RB1 mutation deregulates temporal gene expression profiles of osteoblasts.

  • Heterozygous RB1 mutation in MSCs leads to deregulation of osteogenic genes.

Abstract

Osteosarcoma (OS) is a bone malignancy affecting children and adolescents. Retinoblastoma (RB) patients with germline RB1 mutations are susceptible to osteosarcoma in the second decade of their life. Several studies, particularly in mice, have revealed a role for RB1 in osteogenesis. Since, there is species specific difference attributed in retinoblastoma tumorigenesis between mice and human, we assumed, it is worthwhile exploring the role of RB1 in osteogenesis and thus onset of osteosarcoma. In this study, we analyzed the temporal gene expression of the osteogenic markers, tumor suppressor genes and hormone receptors associated with growth spurt during in vitro osteogenesis of mesenchymal stem cells derived from orbital adipose tissue of germline RB patients and compared it with those with wild type RB1 gene. Mesenchymal stem cells with the heterozygous RB1 mutation showed reduced expression of RB1 and other tumor suppressor genes and showed deregulation of osteogenic markers which could be an initial step for the onset of osteosarcoma.

Introduction

Osteosarcoma (OS) is a rare, but an aggressive malignancy of the bone affecting children and adolescents [1]. Although OS may arise sporadically, a few genetic disorders such as Li Fraumeni syndrome (LFS), hereditary retinoblastoma (RB), Rothmund Syndrome (RTS), Werner syndrome (WS), Bloom syndrome (BS), Paget's disease of the bone (PDB) set a predisposition to osteosarcoma [2,3]. Exome sequencing of osteosarcoma also revealed that mutations in TP53, RB1, BRCA2, ATM, PTEN, RECQL4, BAP1, RET, MUTYH, WRN, ATRX, FANCA, NUMA1 and MDC1 to be drivers or alternate drivers of osteosarcoma [4].

In humans, germline mutation of RB1 results in retinoblastoma and other secondary tumors including osteosarcoma. However, in mice, RB1 mutation alone does not result in either retinoblastoma or osteosarcoma [3,5]. RB1 has been reported to play an important role during osteogenic differentiation. Its role in the transcriptional regulation of osteogenesis by interacting with the master regulator RUNX2 has been demonstrated by earlier studies [6]. Genetically engineered mice carrying osteoblast-specific RB1 mutation had shown osteogenic differentiation defects. In vivo studies have shown that mice lacking RB1 showed defective osteogenesis. In vitro culture of the RB1 deficient osteoblasts showed increased proliferation of the progenitors and reduced gene expression of the osteoblast markers [7,8]. Luan et al., 2007 demonstrated the localisation of the complex (p204-RB protein-RUNX2) on the promoter of the osteoblast marker osteocalcin pointing to role of RB1 in osteogenesis [9]. Deletion of RB1 along with other tumor suppressor PTEN in mice also cause reduced osteogenic gene expression [10]. Loss of RB1 along with other tumor suppressors such as TP53 and PTEN has shown osteogenic defects and features similar to osteosarcoma in mice.

The osteogenic differentiation of the mesenchymal stem cells (MSCs) is shown to be important for the onset of osteosarcoma [11]. Hence, in order to study the role of RB1 in osteosarcoma, it is imperative to know its role in osteogenic differentiation of MSCs. In addition, OS is considered to be a hormonal cancer and occurs mostly during adolescence, which is characterized by rapid skeletal growth with a sudden surge in the insulin-like growth factor (IGF-1), androgen and estrogen [12]. Therefore in this study to gain insight into the role of RB1 in osteogenesis, we compared the osteogenic differentiation between the orbital adipose mesenchymal stem cells (OAMSCs) obtained from unilateral (wild type RB1) and bilateral (heterozygous RB1 mutation) and analyzed the temporal gene expression of osteogenic markers and transcription factors, tumor suppressor genes during the osteogenic differentiation. To understand the role of the growth spurt in osteogenesis we also performed temporal gene expression analysis of hormonal genes such as insulin-like growth factor receptor (IGF1R), Estrogen receptor alpha (ESR1) and Androgen receptor (AR) during the differentiation. Our study reveals that a few genes that were correlated during the differentiation lost its correlation when there was an RB1 mutation.

Section snippets

Ethics approval

The study was approved by Institutional ethics committee and by Institutional committee for stem cell research and therapy under approval numbers 779-2019-P and 18-2019-P, respectively.

Cell culture

Orbital adipose tissue was procured from retinoblastoma patients undergoing therapeutic enucleation after obtaining their informed consent. OAMSCs from the unilateral retinoblastoma patient did not harbor RB1 mutation, served as the wild type. OAMSCs from bilateral retinoblastoma carrying heterozygous RB1

Characterization of OAMSCs

The immunophenotypic characterization of OAMSCs from unilateral and bilateral patients was carried out by assessing the surface marker expression by flow cytometry. The OAMSCs derived from both unilateral as well as the bilateral patients revealed >95% positivity for the adult mesenchymal stem cell markers CD105, CD90 and CD73 (Fig S1-S3). All the OAMSCs showed <2% positivity for the negative markers of MSCs (CD19, HLA-DR, and CD45). Hematopoietic stem cell marker CD34 was positive in

Discussion

The tumor suppressor RB1 is known to play a key role in the differentiation of multiple lineages such as bone, fat, and muscle [13]. Earlier studies have reported that the RB1 protein interacts with transcription factors of different lineage and regulates differentiation [7,14]. RB1 gene upregulation in the osteoblasts and positive correlation with the osteoblast marker observed in our current study also points to the role of RB1 in differentiation. Previously, it has been reported that RB1

Conclusion

Our study shows that RB1 gene expression plays an important role during differentiation as reported by earlier studies. Heterozygous RB1 mutation in the MSCs as seen in the bilateral retinoblastoma patients can cause deregulation of genes during the osteogenic differentiation which could deregulate various pathways of differentiation. To conclude, this patient-derived pluripotent stem cell model serves as an in vitro model to elucidate the initial molecular pathogenesis of osteosarcoma where RB1

Funding

This work was supported by the Indian Council of Medical Research [F. No: 2019–4208/SCR-BMS] and the Department of Biotechnology [grant no: BT/PR 13929/MED/31/304/2015].

CRediT authorship contribution statement

Ambily Vincent: Methodology, Investigation, Validation, Writing - original draft. Viswanathan Natarajan: Investigation, Formal analysis. Vikas Khetan: Resources, Supervision. Subramanian Krishnakumar: Resources, Supervision. Sowmya Parameswaran: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We acknowledge and thank Indian Council of Medical Research (ICMR) for providing the ICMR-Senior Research fellowship to Ms. Ambily Vincent (F.No:2019–4208/SCR-BMS).

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