A subset of HER2+ breast cancer patients manifest clinical resistance to trastuzumab. Recently, miR-26a and miR-30b have been identified as trastuzumab response regulators, and their target gene CCNE2 seems to play an important role in resistance to trastuzumab therapy. Cell viability was evaluated in trastuzumab treated HER2+ BT474 wt (sensitive), BT474r (acquired resistance), HCC1954 (innate resistance), and MDA-MB-231 (HER2−) cell lines, and the expression of miR-26a, miR-30b, and their target genes was measured. BT474 wt cell viability decreased by 60% and miR-26a and miR-30b were significantly overexpressed (~3-fold, p = 0.003 and p = 0.002, respectively) after trastuzumab treatment, but no differences were observed in resistant and control cell lines. Overexpression of miR-30b sensitized BT474r cells to trastuzumab (p = 0.01) and CCNE2, was significantly overexpressed after trastuzumab treatment in BT474r cells (p = 0.032), but no significant changes were observed in sensitive cell line. When CCNE2 was silenced BT474r cell sensitivity to trastuzumab increased (p = 0.03). Thus, the molecular mechanism of trastuzumab action in BT474 cell line may be regulated by miR-26a and miR-30b and CCNE2 overexpression might play an important role in acquired trastuzumab resistance in HER2+ breast cancer given that resistance was diminished when CCNE2 was silenced.

The mechanisms of chemotherapy resistance in triple negative breast cancer remain unclear, and so, new molecules which might mediate this resistance could optimize treatment response. Here we analyzed the involvement of the miRNA-449 family in the response to doxorubicin. The cell viability, cell-cycle phases, and the expression of in silico target genes and proteins of sensitive/resistant triple negative breast cancer cell lines were evaluated in response to doxorubicin treatment and after gain/loss of miRNAs-449 function achieved by transient transfection. Triple negative breast cancer patients were selected for ex vivo experiments and to evaluate gene and miRNAs expression changes after treatment, as well as survival analysis by Kaplan-Meier. Doxorubicin treatment upregulated miRNAs-449 and DNA-damage responder factors E2F1 and E2F3 in triple negative breast cancer sensitive breast cancer cells, while expression remained unaltered in resistant ones. In vitro overexpression of miRNAs-449 sensitized cells to the treatment and significantly reduced the resistance to doxorubicin. These changes showed also a strong effect on cell cycle regulation. Finally, elevated levels of miRNA-449a associated significantly with better survival in chemotherapy-treated triple negative breast cancer patients. These results reveal for the first time the involvement of the miRNA-449 family in doxorubicin resistance and their predictive and prognostic value in triple negative breast cancer patients.

Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis and thereby involved in the development and progression of solid tumours. The association between polymorphisms of angiogenesis pathway genes and risk of breast cancer (BC) has been widely studied, but the results are not conclusive. This information is especially limited in Spanish women, so we decided to conduct a case-control study. Here, we selected four commonly studied polymorphisms in VEGF, rs3025039 (known as +936 C/T), rs1109324, rs154765 and rs833052, one polymorphism at the promoter of the VEGFR1 (-710 C/T) and another in the FGF2, rs1449683, gene to explore their association with BC susceptibility. Genotyping was performed by TaqMan SNP assays and polymerase chain reaction-restriction fragment length polymorphis (PCR-RFLP) on 453 patients and 461 controls in a population from Valencia (Spain). We observed that women carriers of +936 CT + TT VEGF genotypes have a protective effect concerning this disease (p = 0.014; OR 0.67, 95% CI 0.48-0.92) in the global group of patients. The haplotype TGAC of VEGF (rs3025039, rs1109324, rs154764 and rs833052) shows a reduction of the risk to develop BC (p = 3e-04; OR 0.48, 95% CI 0.32-0.72). Furthermore, we found that carriers of -710 CT + TT VEGFR1 genotypes have also a protective effect (p = 0.039; OR 0.55, 95% CI 0.31-0.98). When we stratified by groups of ages these associations were maintained. Our data report for the first time the association of the polymorphism -710 C/T VEGFR1 with BC. Additional experiments focused on VEGF-A, VEGFR1 and sVEGFR1 gene expression demonstrated that carriers of T allele at -710 C/T VEGFR1 genotype have higher levels of sVEGFR1/VEGF-A than the C/C genotype carriers. This was consistent with the hypothesis that this polymorphism may act as low penetrance risk factor. The data provided suggest that +936 C/T VEGF and -710 C/T VEGFR1 genotypes are likely important genetic markers of susceptibility to BC.

No abstract

The tumour-suppressor gene TP53 has been associated with the angiogenic pathway by a TP53 response element sequence of the VEGFR1 promoter. Within that sequence, the polymorphism -710 C/T VEGFR1, which confers variable transcriptional activation by TP53, has been identified. Our group found an association between this polymorphism and breast cancer (BC) risk. We decided to investigate a possible epistatic interaction between this polymorphism and others located at gene TP53. We chose four polymorphisms (Ex4 + 119G > C, IVS4-91A > G, IVS6 + 62A > G and IVS7 + 92T > G) to analyse out of a total of 461 controls and 453 BC patients in a Spanish population. The two-locus combined analysis of TP53 and -710 C/T VEGFR1 polymorphisms was performed with the multifactor dimension reduction approach. Kaplan-Meier disease-free survival curves were calculated using the SPSS package. Carriers of at least one Pro allele of the Ex4 + 119G > C TP53 polymorphism presented a significant BC risk [OR = 1.34, (95 % CI 1.03-1.75), p value = 0.029]. The epistatic gene-gene analysis showed that the best two-locus model was the combination between Ex4 + 119G > C TP53 and -710 C/T VEGFR1 showing OR of 1.44 (95 % CI 1.10-1.88, p value = 0.0083). Moreover, the Pro/Pro genotypes of Ex4 + 119G > C were associated with poor disease-free survival (p value = 0.013). We conclude that the Ex4 + 119G > C TP53 polymorphism is an independent, low penetrance marker of BC risk in this population. In addition, our findings suggest that the combination of Ex4 + 119G > C TP53 and -710 C/T VEGFR1 genotypes confers a higher risk to develop BC. Also, a possible association of the Ex4 + 119G > C TP53 genotype with decreased disease-free survival in these patients is proposed.