Hormonal Carcinogenesis

1) Creation of a Biobank of patient-derived xenografts (PDX) for the development of precision therapeutic strategies in breast cancer

The future of Oncology is based on the development of personalized therapies. Increasingly sophisticated drugs are currently being produced, a technology that must be accompanied by the development of experimental models that allow preclinical testing of their efficacy. The development of PDX is framed in this scenario. The general aim of this project is a) to develop a Biobank of PDX models of breast cancer using traditional methodologies together with newer ones to improve their success, b) to typify PDX from the histological point of view, markers of routine (RE, RP, HER2 and Ki67) and others of our interest and characterize the tumor transcriptome using Exome-seq and RNA-seq; c) validate the use of tissue cultures (breast cancer tissue cultures; BCTC) developed from PDX (BCTC-PDX) to test the effect of different combinations of drugs that will be chosen according to the molecular profile of each PDX and then use the most relevant therapies selected from in vitro studies, in in vivo experiments and d) develop models of acquired resistance in PDX already achieved in the laboratory to offer resistance models for testing the efficacy of second or third therapies line. The PDX models achieved will serve a) to strengthen and enhance the developments of other researchers in our country, b) offer a local product to biotechnological or pharmaceutical companies that need to test their biotechnological developments in preclinical studies and c) eventually they can be exported directly or offered to license their sale in foreign biotechnological companies that are dedicated to carrying out these tests on request, or offered at IBYME to carry out biotechnological studies with a high-tech service (STAN) from CONICET.

2) Clinical study: Mifepristone treatment for breast cancer patients with higher expression levels of progesterone receptor isoform A than isoform B

Based on the background of our laboratory of work carried out over the last twenty years, we designed a clinical study to treat breast cancer patients with levels of PR isoform A higher than isoform B, with mifepristone, for 14 days, between biopsy and surgery (MIPRA; NCT02651844). The primary outcome was to compare proliferation markers before and after mifepristone treatment. It was carried out in collaboration with Hospital Magdalena V de Martínez de General Pacheco, Buenos Aires. In addition to the primary endpoint, morphological, transcriptomic and proteomic studies were carried out to validate the data. Based on these results, we are now studying differential markers that may be useful for selecting patients who will respond to this treatment.

3) Vulnerability conditions and their relationship with the stage of presentation at the first consultation in patients with breast cancer who attend the Magdalena V de Martínez Hospital in General Pacheco.

Breast cancer is the most frequent oncological disease in women, and one of the main causes of death. Early detection through breast examination and mammography is the best strategy for reducing mortality. The difficulty of access to timely evaluation, due to lack of knowledge or institutional barriers, constitutes the main factor of vulnerability in the population. This study aims to identify, in patients with advanced breast cancer, the factors for which they did not attend the hospital and, consequently, the corresponding studies were not performed, in a timely manner. This is an observational study (exploratory-descriptive or correlational, prospective, cross-sectional and mixed). Based on the results obtained in this project, we will be able to: 1) develop plans to improve prevention, 2) optimize the time that elapses between the first consultation and the start of treatment and, 3) work together with the local authorities in educational and preventive campaigns, so that patients go to the hospital in early stages of breast cancer.

4) Combination therapies with progesterone receptor ligands in breast cancer.

Seventy percent of mammary carcinomas express estrogen receptor alpha (ER) and progesterone receptor (PR) and are sensitive to endocrine therapies. There are two isoforms of PR, PRB corresponds to the complete sequence, and PRA, to a truncated protein. We postulate that patients with tumors expressing higher levels of PRA than PRB (PRA-H) may benefit from treatment with antiprogestagens such as mifepristone (MFP), while those expressing higher levels of PRB than PRA (PRB-H) could benefit from progesin therapy. There are currently clinical trials with both types of ligands. Only our MIPRA clinical trial discriminates patients according to the ratio of PR isoforms to treat them with MFP (NCT02651844). Taking into account that three CDK4/6 inhibitors have recently been approved for the treatment of advanced breast cancer in conjunction with endocrine therapy, we intend to explore the effects of combined therapies vs single therapies of the CDK4/6 inhibitor Palbocilcib (PALBO) with MFP in PRA-H scenarios or with progestins in PRB-H. We will use three families of murine mammary carcinomas that have PRA-H and PRB-H variants, from the model known as “”The MPA-induced breast cancer model””, developed by our group. We have already sequenced the exome (ExomeSeq) to find out the mutation profile of the C7 family, and in this project we will study the other two families C4 and 59. The effects of simple and combined therapies will be evaluated in each of them. Human T47D cells using the variants that overexpress one or another isoform of PR will also be used. In vitro, it will be analyzed how therapeutic agents regulate cell proliferation, the expression and activation of proteins related to the cell cycle. In xenografts of these modified cell lines, growing in NSG mice, responses on tumor growth and metastasis will be evaluated. In PDX and in tissue cultures (BCTC) of these luminal PDX, the in vivo response will be evaluated and the response will be associated with the PR isoform profile. Finally, in BCTC performed from patient samples cataloged by their PR isoform profile, the ex vivo response will be evaluated. With the information obtained from the sequencing of the exome of the murine model, and from the regulation of expression of cell cycle proteins studied in in vitro assays in this project, and confirmed in tissue sections of the in vivo models, possible candidate genes will be selected to help us to discriminate patients in whom the response to combined therapies with PR ligands and CDK4/6 inhibitors could be predicted. If our goals are achieved, a new arsenal will be available for luminal breast cancer patients discriminated by the ratio of PR isoforms.

5) Activation of the Wnt pathway and androgen receptors induced by FGF2: involvement in the progression of breast cancer

We hypothesize that in endocrine-resistant breast carcinomas, increased intracellular high molecular weight (HMW)-FGF2 contributes to the activation of the Wnt pathway, which, in turn, regulates the expression of transcription factors capable of modulating androgen receptor expression (AR) and PR. The decrease in PR levels together with the increase in AR levels, would rank the role of AR in tumor progression, positioning treatment with antiandrogens (AA) as a therapeutic alternative in tumors resistant to antiprogestin therapy (AP). Our objective is to elucidate the mechanisms involved in FGF2-induced tumor progression, determining the participation of the Wnt pathway in the regulation of the positive expression of AR and the negative regulation of PR. For this, we will use experimental models that express ER, low levels of PR and high levels of AR, which would make up a subgroup of luminal tumors represented in the clinic. We postulate that this group of patients could benefit from therapy with anti-androgens and/or inhibitors of the Wnt pathway, which, in turn, restores endocrine sensitivity, and allows them access to a more specific therapy for this tumor subtype.

6) Study of the participation of cell cycle proteins in tumor progression of different experimental models of breast cancer

Most human breast cancers have some of the proteins that control cell division deregulated. In particular, the expression of cyclin A (which regulates the progression of the S and G2-M phases of the cell cycle) would be associated with prognosis in breast cancer. On the other hand, cyclin A could act as a coactivator of the progesterone receptor (PR), activating genes induced by progestins. The study of the expression of cyclin A in the murine model of breast cancer induced by progestins revealed a possible role of cyclin A in the mechanism of resistance to antiprogestagens. In addition, an association was observed between cyclin A expression levels and PR isoforms in a model of human breast cancer. The general aim of the project is to evaluate the participation of the cyclin A/CDK2 complex in tumor progression and in the response to endocrine therapy in different experimental models of breast cancer. To this end, cyclin expression was silenced in the T47D model and the response to progestins and antiprogestins, both in vitro and in vivo, is being evaluated. The participation of the cyclin A-associated kinase, CDK2, in the endocrine response using the inhibitor roscovitine is also being analyzed. In addition, the interaction of cyclin A with the different PR isoforms and the regulation of gene transcription induced by progestins/antiprogestins in the different experimental models is being studied.