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ETV7 Regulates Breast Cancer Stem-Like Cell Plasticity by Repressing IFN- Response Genes

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ETV7 Regulates Breast Cancer Stem-Like Cell Plasticity by Repressing IFN- Response Genes bioRxiv preprint doi: https://doi.org/10.1101/2020.09.02.279133; this version posted September 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 ETV7 regulates breast cancer stem-like cell plasticity by repressing IFN- 2 response genes 3 4 5 6 7 Laura Pezzè1,#, Mattia Forcato2, Stefano Pontalti1,^, Kalina Aleksandra Badowska1,#, Dario 8 Rizzotto3,°, Ira-Ida Skvortsova4,5, Silvio Bicciato2, Yari Ciribilli1,§ 9 10 11 12 1 Laboratory of Molecular Cancer Research, Department of Cellular, Computational and Integrative 13 Biology (CIBIO), University of Trento, Italy; 2 Department of Life Sciences, University of Modena 14 and Reggio Emilia, Italy; 3 Laboratory of Transcriptional Networks, Department CIBIO, University 15 of Trento, Italy; 4 EXTRO-Lab, Department of Therapeutic Radiology and Oncology, Medical 16 University of Innsbruck, Austria; 5 Tyrolean Cancer Research Institute, Innsbruck, Austria 17 # present address: Alia Therapeutics s.r.l., Trento, Italy 18 ^ present address: Azienda Provinciale per i Servizi Sanitari, APSS, Trento, Italy 19 °present address: CeMM Research Center for Molecular Medicine of the Austrian Academy of 20 Sciences, Vienna, Austria 21 § correspondence: [email protected] (YC) 22 23 24 25 Running Title: ETV7 regulates BCSCs plasticity 26 27 28 Keywords: ETV7 – Cancer stem cells – Breast cancer – Interferon – Chemoresistance 29 30 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.02.279133; this version posted September 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 31 ABSTRACT 32 33 Cancer stem cells (CSCs) represent a population of cells within the tumor able to drive 34 tumorigenesis and known to be highly resistant to conventional chemotherapy and radiotherapy. In 35 this work, we show a new role for ETV7, a transcriptional repressor member of the ETS family, in 36 promoting breast cancer stem-like cells plasticity and resistance to chemo- and radiotherapy in 37 breast cancer (BC) cells. We observed that MCF7 and T47D BC-derived cells stably over- 38 expressing ETV7 showed reduced sensitivity to the chemotherapeutic drug 5-Flouororuacil and to 39 radiotherapy, accompanied by an adaptive proliferative behavior observed in different culture 40 conditions. We further noticed that alteration of ETV7 expression could significantly affect the 41 population of breast CSCs, measured by CD44+/CD24low cell population and mammosphere 42 formation efficiency. By transcriptome profiling, we identified a signature of Interferon-responsive 43 genes significantly repressed in cells over-expressing ETV7, which could be responsible for the 44 increase in the breast CSCs population, as this could be partially reverted by the treatment with 45 IFN-β. Lastly, we show that the expression of the IFN-responsive genes repressed by ETV7 could 46 have prognostic value in breast cancer, as low expression of these genes was associated with a 47 worse prognosis. Therefore, we propose a novel role for ETV7 in breast cancer stem cells’ plasticity 48 and associated resistance to conventional chemotherapy and radiotherapy, which involves the 49 repression of a group of IFN-responsive genes, potentially reversible upon IFN-β treatment. We, 50 therefore, suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs 51 targeted therapies and to the improvement of combinatorial regimens, possibly involving the 52 therapeutic use of IFN-β, with the aim of avoiding resistance development and relapse in breast 53 cancer. 54 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.02.279133; this version posted September 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 55 INTRODUCTION 56 57 Breast cancer is the most frequent tumor and the leading cause of cancer-related deaths in women 1. 58 Breast cancer is a highly genetically heterogenous disease 2, and current therapeutic approaches are 59 chosen based on the subtype of cancer, stage, mass and localization of the tumors among several 60 parameters 3. The most common therapeutic strategies rely both on local (e.g., surgery and 61 radiotherapy) and systemic treatments (e.g., chemotherapy, endocrine/hormonal and targeted 62 therapy) 4. Hormone-dependent breast cancers, such as luminal breast cancers, frequently become 63 refractory to the initially effective hormonal treatments, thus eventually requiring chemotherapy 5. 64 Chemotherapy, in fact, still represents the most common option for advanced breast cancers. 65 However, it has been demonstrated that chemotherapy itself, despite triggering shrinkage of 66 primary tumors, can enhance metastasis through induction of EMT 6. Moreover, cancer cells can 67 develop drug resistance, resulting in treatment failure and recurrence 7, 8. Cancer stem cells (CSCs) 68 are considered the population of cells within the tumor able to drive tumorigenesis. CSCs are 69 characterized by self-renewal ability and differentiation potential, and can therefore contribute to 70 the heterogenicity of cancer cells within the tumor 9, 10. Moreover, they are known to be intrinsically 71 highly resistant to conventional anti-cancer agents, including chemotherapy and radiotherapy 11, 12. 72 Therefore, uncovering the pathways and mechanisms involved in CSCs enrichment, development of 73 drug resistance or other unwanted side effects associated with chemotherapy is an urgent and 74 critical aim for cancer research, oriented to improve treatment efficacy. 75 Using genome-wide transcriptional profiling of MCF7 human breast adenocarcinoma-derived cells, 76 we identified ETV7 as a potential tumor-promoting factor among the genes more synergistically up- 77 regulated by the combinatorial treatment with the chemotherapeutic agent Doxorubicin and the 78 inflammatory cytokine TNFα 13. 79 ETV7, also called TEL2 or TELB, is a member of the large family of ETS (E26 Transforming 80 Specific) transcription factors, whose members can regulate the expression of genes involved in 81 various processes, such as development, differentiation, cell proliferation, migration, and apoptosis 82 14, 15. Given their role in essential cellular functions, their dysregulation can result in severe 83 impairments within the cell, as demonstrated by the involvement of ETS factors in various diseases 84 16-18. In particular, many ETS factors have been associated with cancer initiation, transformation 85 and metastatic spread 19-22. The roles of ETV7 are still poorly understood and studied, partially 86 because of the absence of ETV7 gene in most rodent species, including mice 23. However, several 87 studies highlighted multiple roles for ETV7 in hematopoiesis. For examples, the over-expression of 88 ETV7 in both human and mice hematopoietic stem cells (HSCs) could increase the proliferation 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.02.279133; this version posted September 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 89 and deplete HSCs 24. Moreover, ETV7 over-expression in human U937 cells was shown to impede 90 monocytic differentiation 25. ETV7 is also recognized as an Interferon (IFN)-stimulated gene (ISG), 91 as its expression was shown to be induced by type I, type II and type III IFN treatment in different 92 cell types 26-28. Elevated ETV7 expression has been associated with several tumor types; however, 93 the role of ETV7 in cancer has been poorly investigated. ETV7 was shown to cooperate with Eμ- 94 MYC in promoting B-lymphomagenesis 29 and the forced expression of ETV7 in mouse bone 95 marrow was shown to cause myeloproliferative diseases, even if with a long latency, suggesting 96 TEL2 as a bona fide oncogene 30. Besides, the crossing of the latest established ETV7 transgenic 97 mouse model with an established leukemic mouse model revealed a remarkable acceleration in 98 Pten-/- leukemogenesis 31. Further evidence supporting ETV7 pro-tumorigenic functions comes 99 from the recent identification of a transcriptional-independent activity of ETV7, which was shown 100 to physically interact with mTOR into the cytoplasm generating a novel complex called mTORC3, 101 which contributes to resistance to rapamycin, an mTOR-targeting anti-cancer agent 32. In contrast 102 with these observations, ETV7 was shown to act as tumor suppressor in nasopharyngeal carcinoma 103 by repressing SERPINE1 33, and its down-regulation was observed in drug-resistant cancer cells 34. 104 Recently, Piggin and colleagues reported an average higher level of ETV7 expression in tissues 105 from all the breast cancer subtypes compared to normal breast tissues, with a correlation of ETV7 106 expression and breast cancer aggressiveness 35. We have recently demonstrated in breast cancer- 107 derived cells that ETV7 expression can be induced by DNA-damaging chemotherapy, and elevated 108 ETV7 levels has been associated with reduced sensitivity to Doxorubicin treatment, a mechanism 109 which involve ETV7-mediated direct repression of DNAJC15, resulting in the increase in ABCB1 110 expression levels 36. 111 In this study we further demonstrated the role of ETV7 in breast cancer cell resistance to another 112 chemotherapeutic drug, 5-Fluorouracil (5-FU) and radiotherapy. Noteworthy, we were able to 113 observe a remarkable increase in breast cancer stem cells content in the cells over-expressing 114 ETV7. We finally identified via RNA-seq a set of type I and type II Interferon response genes that 115 were repressed by ETV7 as putative responsible of the stem cells enrichment.
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