Mecanismos involucrados en la resistencia hormonal de carcinomas mamarios humanos: participación de las isoformas del factor de crecimiento fibroblástico 2 (FGF2)
Seventy-five per cent of mammary carcinomas express estrogen receptor alpha (ER) and progesterone receptors (PR) and are susceptible to endocrine therapy. However, over time, many of these patients develop resistance to therapy. In our laboratory, using murine and human mammary carcinoma models, we...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica
2022
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=posgraafa&cl=CL1&d=HWA_6735 https://repositoriouba.sisbi.uba.ar/gsdl/collect/posgraafa/index/assoc/HWA_6735.dir/6735.PDF |
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| Sumario: | Seventy-five per cent of mammary carcinomas express estrogen receptor alpha (ER) and progesterone receptors (PR) and are susceptible to endocrine therapy. However, over time, many of these patients develop resistance to therapy. In our laboratory, using murine and human mammary carcinoma models, we have shown that tumors that respond to therapy with the antiprogestin mifepristone (MFP) have higher expression of PR isoform A (PRA) than isoform B (PRB), while those with higher levels of PRB than PRA are MFP-resistant tumors.\nVarious hypotheses have been proposed to explain the mechanisms leading to endocrine resistance, including constitutive activation of signal transduction pathways. The FGF2/FGFR pathway involves four receptors (FGFR1-4) and five FGF2 isoforms with different molecular weights: the 18-kDa isoform or LMW-FGF2, and the high molecular weight (HMW)-FGF2 of 22-, 22,5, 24 and 34 kDa. In previous studies we demonstrated that, in MFP-responsive tumors, FGFR2 activated by stromal FGF2 participates in tumor growth through PR activation. Likewise, in murine and human experimental models, we demonstrated that endocrine-resistant mammary carcinomas express higher levels of intracellular HMW-FGF2 than responsive variants and that HMW-FGF2 overexpression induces endocrine resistance and an increase in metastatic spread. Although it has been shown that the FGF2/FGFR pathway is deregulated in numerous neoplasms and developmental pathologies, in breast cancer, many questions remain to be answered regarding the role of FGF2 isoforms and the mechanisms involved in the acquisition of FGF2-induced endocrine resistance. Moreover, the studies that report the expression of total FGF2 in samples from patients with breast cancer are still controversial, and there are no studies that investigate whether there is an association between the expression of FGF2 isoforms and disease prognosis.\nThus, our working hypothesis is that in the T47D cell line, the overexpression of LMW- or HMW-FGF2 isoforms differentially regulates the activation of signaling pathways, which include crosstalk with hormone receptor pathways, and that favors tumor growth and progression. Therefore, our main aim was to evaluate the role of the different FGF2 isoforms in luminal breast cancer progression and the association with hormone receptor pathways.\nIn the first place, we used the cell lines already available in our laboratory, T47D-YA-p6NST50, -YA-18 and -YA-22,5, which express only PRA, and also express an empty plasmid, or plasmids containing the 18 kDa or the 22,5 kDa isoforms of FGF2. We performed RNA-seq assays to identify differentially expressed transcripts in the presence of one or another FGF2 isoform to find candidates involved in tumor progression induced by these isoforms. We observed that, in both cell lines, FGF2 overexpression induces the deregulation of different pathways, like the Wnt pathway and the expression of hormone receptors, among others, that could affect tumor progression. Although both -LMW and -HMW overexpression induce deregulation of the Wnt pathway, we observed a differential deregulation in WNT ligands. Furthermore, we determined that both FGF2 variants induce a decrease in ESR1 and PGR along with an increase in AR transcription levels.\nWe then developed a model of FGF2 isoform overexpression that would better reflect what occurs in breast cancer patients that carry PR+ tumors which normally express both PR isoforms. Therefore, we stably transfected the T47D cell line with the plasmids containing the 18 kDa or 22 kDa FGF2-isoforms. Upon characterization we observed that, as in the T47D-YA model, LMW- and HMW-FGF2-overexpression favors tumor progression determined as an increase in tumor growth, metastatic dissemination, and the acquisition of antiprogestin resistance. Next, we performed Western blot and RT-qPCR assays to determine hormone receptor expression and we found that the overexpression of both FGF2 isoforms, in the T47D and in the T47D-YA cell lines, induces the decrease of ER and PR and, in the case of the T47D-18 and -22 lines, this decrease in PR was more pronounced for PRA than PRB resulting in a low PRA/PRB ratio, which is consistent with an endocrine resistant phenotype, according to previous results from our lab. These results would allow to explain the acquisition of MFP resistance investigated in depth in other previous studies from our laboratory. That is, the increase in total FGF2 could be favoring the PRB phenotype and, therefore, play an important role in antiprogestin resistance. Furthermore, we determined that, in the T47D model, only HMW-FGF2, and not LMW-FGF2, induced an increase in AR expression. The differences between the cell lines that overexpress one or another FGF2 isoform were also reflected in a differential response to agonists or antagonists of ER, PR, AR and inhibitors of the Wnt pathway, as well as in the activation of the AKT pathway, which reinforces the idea that both isoforms would have differential roles.\nNext, we investigated the cross-regulation of FGF2, PR, ER, and AR expression. To this end, we used conditioned media from cell lines that overexpress FGF2 isoforms to investigate whether they were capable of modulating ER and PR expression. We observed that the decrease in ER observed in both T47D-18 and -22 would be an indirect effect of FGF2 action, which would induce the secretion of soluble factors such as WNT4, which, in turn, trigger other signals that impact ER expression. On the other hand, we determined that FGF2 induces PR decrease through a different and ER-independent mechanism.\nMoreover, to determine whether PRB or AR can regulate the FGF2 expression, we transfected the T47D-Y cell line, which does not express PR, and the T47D-YA with a constitutive expression plasmid of PRB and found an increase in FGF2 expression. Then, we designed a reporter plasmid containing the FGF2 promoter coupled to the luciferase gene, transfected the T47D-YB cell line, and evaluated luciferase expression in the presence of progestogens, androgens, and antiandrogens. We observed that progestins repressed FGF2 promoter activity, which is consistent with the inhibitory effect of progestins in a PRB context. In addition, we found an effect in the same direction when treating with an antiandrogen, which suggests that both PRB and AR would be basally active in a ligand-independent manner and inducing FGF2.\nThe fact that the T47D-22 cell line has a deregulated Wnt pathway and an increased AR expression positioned it for treatment with inhibitors of both pathways. We carried out in vitro and in vivo assays and found that this tumor is sensitive to treatment with both inhibitors. In addition, we determined that combined treatment with these drugs decreases not only tumor growth but also the dissemination and growth of metastatic foci.\nFinally, to study whether there is a correlation between the nuclear expression of FGF2 and AR, we evaluated the expression and localization of both proteins in breast cancer samples from the Magdalena V. de Martínez hospital, focusing on luminal tumors. We observed that patients with higher expression of nuclear FGF2 also express a higher percentage of AR. When we classified the samples by tumor subtype, we observed a similar association in HER2+ tumors, and a trend in the same direction is even distinguished in the ER+PR- subgroup, which is the subgroup that most resembles our experimental model. These results suggest that there is an association between the AR and FGF2 pathways.\nTogether, in this work we demonstrate that the LMW- and HMW-FGF2 isoforms have differential roles and favor tumor progression in a luminal model of breast cancer. Our results suggest that ligand-independent activation of PRB or AR would induce the expression of FGF2, which in turn would increase the secretion of Wnt ligands, favoring tumor progression.\nWe propose that the subgroup of ER+PR-AR+ tumors with nuclear expression of FGF2 could benefit from an antiandrogenic therapy combined with inhibitors of the Wnt pathway. This therapy could even benefit patients with advanced breast cancer. |
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