Final Report Summary - TNBCHEMIRNET (Enhancing chemotherapy response in triple negative breast cancer (TNBC) by modulating miRNA-target network and identifying biomarkers of response)
Triple negative breast cancer (TNBC) is one of the most aggressive cancers with lack of targeted therapy options. Currently, chemotherapy is the mainstay treatment for TNBC patients; however, development of resistance is a major obstacle in clinics whose underlying molecular mechanisms are poorly understood. In this project, we hypothesized that miRNA/target networks might play key roles in chemoresistance, and miRNAs can be utilized to predict and enhance chemoresponse. To test these, we aimed to 1.) develop chemoresistant TNBC models, 2.) perform RNA/miRNA Sequencing to identify miRNA/target networks involved in chemoresistance, 3.) target miRNAs or mRNA targets to prevent/delay chemoresistance, and 4.) test biomarker potential of miRNAs/targets to predict chemoresponse in TNBC patients.
To elucidate the mechanisms of chemoresistance in TNBCs, we developed doxorubicin-resistant TNBC tumors in nude mice. We treated tumor-bearing mice with doxorubicin and collected “sensitive” and “resistant” tumors depending upon their response over time. We complemented this model with developing an in vitro 3D culture TNBC chemoresistance model. We performed RNA and miRNA sequencing to find the differentially expressed genes between sensitive and resistant tumors. To construct miRNA/target network, we used Ingenuity Pathway Analysis (IPA) to determine interactions between the network components and added transcription factors regulating miRNAs as the connectors in the network. We refined this network by considering the effect of mRNA expression on chemotherapy-treated TNBC patient survival. We computationally identified the key pathways involved in chemoresistance. We experimentally tested the effects of three candidate miRNAs and three mRNAs both in vitro and in vivo, where we showed that miRNAs act as chemosensitizers and their target mRNAs lead to chemoresistance. Finally, we investigated the biomarker potential of these miRNAs and mRNAs in chemotherapy-treated TNBC patients.
As response to chemotherapy strongly correlates with TNBC patients’ survival, increasing the pCR rate to chemotherapy is expected to significantly impact clinical outcome. The outcomes of this project have potential to stratify TNBC patient subpopulations with higher likelihood of chemoresponse, which in turn can increase the survival of patients and save the tax-payers’ money. This is the first study which delineated chemoresistance in TNBC at miRNA-mRNA network level. This led us to identify three major miRNAs and three key proteins, and their modulation could overcome chemoresistance. Importantly, inhibitors or antibodies, which are already available against the proteins we have identified in this project, can in future be translated into the clinics. Furthermore, targetome of the miRNAs in the network may lead us to develop combinatorial therapies to simultaneously block several oncogenic pathways. A rapid translation to clinics could be envisioned by initiating clinical trials combining chemotherapy with the identified combinatorial therapies.
This MC-CIG grant has already resulted in one direct publication in an international journal (Raza et al., 2016, Oncotarget), and currently we are preparing two more direct publications to be submitted soon (Saatci et al, and Assidicky et al,). In addition to these three manuscripts, there is one indirect manuscript (in a collaborative TNBC project with University of Erlangen, GERMANY) recently submitted to the journal Cell Death and Differentiation. During this project, one PhD and two Master students were supported and/or trained, and two of them (Umar Raza and Erol Eyupoglu) obtained their graduate degrees. These students learned how to develop in vivo chemoresistant animal models, preparing samples for RNA/miRNA sequencing, loss-of-function experiments with siRNAs, shRNAs, CRISPR constructs, culturing cells in 3D culture, etc. Furthermore, they gained experience with using different bioinformatics tools. e.g. how to analyze miRNA/RNA-Seq data, construction of networks and visualization, CRISPR guide RNA design etc. Overall, this MC-CIG achieved its aims: 1.) The PI was re-integrated to the European research community; 2.) He transferred the knowledge to his peers and trainee; and 3.) He disseminated the findings to international research community via several international seminars/talks and papers/manuscripts.
The continuously updated project website is available at http://ozgursahinlab.com/mc-cig-tnbchemirnet/
To elucidate the mechanisms of chemoresistance in TNBCs, we developed doxorubicin-resistant TNBC tumors in nude mice. We treated tumor-bearing mice with doxorubicin and collected “sensitive” and “resistant” tumors depending upon their response over time. We complemented this model with developing an in vitro 3D culture TNBC chemoresistance model. We performed RNA and miRNA sequencing to find the differentially expressed genes between sensitive and resistant tumors. To construct miRNA/target network, we used Ingenuity Pathway Analysis (IPA) to determine interactions between the network components and added transcription factors regulating miRNAs as the connectors in the network. We refined this network by considering the effect of mRNA expression on chemotherapy-treated TNBC patient survival. We computationally identified the key pathways involved in chemoresistance. We experimentally tested the effects of three candidate miRNAs and three mRNAs both in vitro and in vivo, where we showed that miRNAs act as chemosensitizers and their target mRNAs lead to chemoresistance. Finally, we investigated the biomarker potential of these miRNAs and mRNAs in chemotherapy-treated TNBC patients.
As response to chemotherapy strongly correlates with TNBC patients’ survival, increasing the pCR rate to chemotherapy is expected to significantly impact clinical outcome. The outcomes of this project have potential to stratify TNBC patient subpopulations with higher likelihood of chemoresponse, which in turn can increase the survival of patients and save the tax-payers’ money. This is the first study which delineated chemoresistance in TNBC at miRNA-mRNA network level. This led us to identify three major miRNAs and three key proteins, and their modulation could overcome chemoresistance. Importantly, inhibitors or antibodies, which are already available against the proteins we have identified in this project, can in future be translated into the clinics. Furthermore, targetome of the miRNAs in the network may lead us to develop combinatorial therapies to simultaneously block several oncogenic pathways. A rapid translation to clinics could be envisioned by initiating clinical trials combining chemotherapy with the identified combinatorial therapies.
This MC-CIG grant has already resulted in one direct publication in an international journal (Raza et al., 2016, Oncotarget), and currently we are preparing two more direct publications to be submitted soon (Saatci et al, and Assidicky et al,). In addition to these three manuscripts, there is one indirect manuscript (in a collaborative TNBC project with University of Erlangen, GERMANY) recently submitted to the journal Cell Death and Differentiation. During this project, one PhD and two Master students were supported and/or trained, and two of them (Umar Raza and Erol Eyupoglu) obtained their graduate degrees. These students learned how to develop in vivo chemoresistant animal models, preparing samples for RNA/miRNA sequencing, loss-of-function experiments with siRNAs, shRNAs, CRISPR constructs, culturing cells in 3D culture, etc. Furthermore, they gained experience with using different bioinformatics tools. e.g. how to analyze miRNA/RNA-Seq data, construction of networks and visualization, CRISPR guide RNA design etc. Overall, this MC-CIG achieved its aims: 1.) The PI was re-integrated to the European research community; 2.) He transferred the knowledge to his peers and trainee; and 3.) He disseminated the findings to international research community via several international seminars/talks and papers/manuscripts.
The continuously updated project website is available at http://ozgursahinlab.com/mc-cig-tnbchemirnet/