DMBA induced mouse mammary tumors display high incidence of activating Pik3caH1047 and loss of function Pten mutations.

Controversy always existed on the utility of chemically induced mouse mammary carcinogenesis models as valid equivalents for the study of human breast cancer. Here, we performed whole exome and RNA sequencing on long latency mammary tumors (218 ± 27 days) induced by the carcinogen 7,12-Dimethylbenzathracene (DMBA) and short latency tumors (65 ± 11 days) induced by the progestin Medroxyprogesterone Acetate (MPA) plus DMBA in CD2F1 mice. Long latency tumors displayed a high frequency of Pi3kca and/or Pten mutations detected in 11 of 13 (85%) long latency cases (14/22, 64% overall). Eighty-two percent (9/11) of tumors carried the Pik3ca H1047L/R hot-spot mutation, as frequently found in human breast cancer. These tumors were luminal-like and mostly ER/PR+, as in humans. Transcriptome profiling indicated a significant activation of the PI3K-Akt pathway (p=3.82e-6). On the other hand MPA+DMBA induced short latency tumors displayed mutations in cancer drivers not commonly found mutated in human breast cancer (e.g. Hras and Apc). These tumors were mostly basal-like and MPA exposure led to Rankl overexpression (60 fold induction) and immunosuppressive gene expression signatures. In summary, long latency DMBA induced mouse mammary tumors reproduce the molecular profile of human luminal breast carcinomas representing an excellent preclinical model for the testing of PIK3CA/Akt/mTOR pathway inhibitory therapies and a good platform for the developing of additional preclinical tools such as syngeneic transplants in immunocompetent hosts.

A Molecular Portrait of High-Grade Ductal Carcinoma In Situ.

Ductal carcinoma in situ (DCIS) is a noninvasive precursor lesion to invasive breast carcinoma. We still have no understanding on why only some DCIS lesions evolve to invasive cancer whereas others appear not to do so during the life span of the patient. Here, we performed full exome (tumor vs. matching normal), transcriptome, and methylome analysis of 30 pure high-grade DCIS (HG-DCIS) and 10 normal breast epithelial samples. Sixty-two percent of HG-DCIS cases displayed mutations affecting cancer driver genes or potential drivers. Mutations were observed affecting PIK3CA (21% of cases), TP53 (17%), GATA3 (7%), MLL3 (7%) and single cases of mutations affecting CDH1, MAP2K4, TBX3, NF1, ATM, and ARID1A. Significantly, 83% of lesions displayed numerous large chromosomal copy number alterations, suggesting they might precede selection of cancer driver mutations. Integrated pathway-based modeling analysis of RNA-seq data allowed us to identify two DCIS subgroups (DCIS-C1 and DCIS-C2) based on their tumor-intrinsic subtypes, proliferative, immune scores, and in the activity of specific signaling pathways. The more aggressive DCIS-C1 (highly proliferative, basal-like, or ERBB2(+)) displayed signatures characteristic of activated Treg cells (CD4(+)/CD25(+)/FOXP3(+)) and CTLA4(+)/CD86(+) complexes indicative of a tumor-associated immunosuppressive phenotype. Strikingly, all lesions showed evidence of TP53 pathway inactivation. Similarly, ncRNA and methylation profiles reproduce changes observed postinvasion. Among the most significant findings, we observed upregulation of lncRNA HOTAIR in DCIS-C1 lesions and hypermethylation of HOXA5 and SOX genes. We conclude that most HG-DCIS lesions, in spite of representing a preinvasive stage of tumor progression, displayed molecular profiles indistinguishable from invasive breast cancer.

Multidrug resistance modulators PSC 833 and CsA show differential capacity to induce apoptosis in lymphoid leukemia cell lines independently of their MDR phenotype.

Among the mechanisms that induce multidrug resistance (MDR), one of those most frequent is over-expression of a phosphoglycoprotein (Pgp) encoded in the mouse by the mdr-1 and mdr-3 genes. We have demonstrated that cyclosporin-A (CsA) as well as its analogue PSC 833 were able to revert the MDR phenotype in murine cell lines resistant to vincristine (LBR-V160) or doxorubicin (LBR-D160). The aim of this work was to evaluate the ability of PSC 833 and CsA to modulate mdr-1, mdr-3 and mrp-1 genes as well as to induce apoptosis analyzing the mechanism involved in the above tumor cell lines. By semi-quantitative RT-PCR, we demonstrated that mdr-3 was over-expressed in both resistant lines while mdr-1 was over-expressed only in LBR-V160; in contrast, mrp-1 expression was not evidenced in any of the cell lines. After treatment with 0.1 microg ml(-1) of either PSC 833 or CsA, LBR-V160 showed no changes in mdr-1 but decreased mdr-3 expression, while LBR-D160 failed to display any modification in the expression of these genes. Apoptosis was evidenced by fluorescence microscopy, S minuscule accumulation and agarose gel electrophoresis. Our results demonstrated that CsA (1 microg ml(-1)) was able to induce apoptosis in all cell lines: 18.31% (+/-4.46) for LBR-, 25.96% (+/-5.24) for LBR-V160 and 27.36% (+/-4.12) for LBR-D160, while PSC 833 (1 microg ml(-1)) only induced apoptosis 21.51% (+/-5.73) in LBR-V160 cell line. The expression of Bcl-2 family proteins (Bcl-2, Bax and Bcl-x(L)) was analyzed by flow cytometry showing high expression of the three proteins which was not significantly modified after treatment with either PSC 833 or CsA on the sensitive as well as on the resistant cell lines. Single stranded conformation polymorphisms analysis of p53 (Trp53) gene in the cell lines showed no mutation in exons 5-8 of the tumor suppressor gene. We conclude that depending on the concentration used, PSC 833 and CsA may act either by modulating the mdr-3 gene (0.1 microg ml(-1)) or by direct impact on the cells through induction of apoptosis (1 microg ml(-1)), in the latter case through a mechanism that might act independent of the Bcl-2 family proteins.

p21, p27 and p53 in estrogen and antiprogestin-induced tumor regression of experimental mouse mammary ductal carcinomas.

Metastatic mammary carcinoma tumor lines 59-2-HI and C7-2-HI originated in female BALB/c mice treated with medroxyprogesterone acetate and are maintained by syngeneic transplantation. Both lines express estrogen (ER) and progesterone receptors (PR) and regress completely after estradiol (E(2)) or antiprogestin treatment. The BET tumor line, of similar origin and biological features, regresses only after E(2) treatment. To investigate possible differences between E(2)- and antiprogestin-mediated effects we evaluated the morphological features, mitosis and apoptosis, and the differential expression of cell-cycle inhibitors associated with tumor regression. Treatments started when tumors reached 50-100 mm(2). After 24-96 h, tumors were excised and processed for morphological and immunohistochemical studies. Regression was associated with a significant and early decrease in the number of mitosis and with higher percentages of apoptotic cells. These phenomena were accompanied by an increase in p21 and p27 expression in the E(2) and antiprogestin-responsive lines treated with E(2), RU 38.486 or ZK 98.299 (P < 0.05). In BET tumors treated with E(2), p21 expression remained within basal levels and only p27 increased (P < 0.05). p53 was low in control 59-2-HI and C7-2-HI tumors and increased after treatment (P < 0.05) whereas BET untreated tumors already expressed high levels of p53 and MDM2. Although the immunohistochemical findings were compatible with alterations of p53, SSCP evaluation failed to disclose the presence of mutations, suggesting that the defective expression of p21 is related to an impaired p53 pathway. UV irradiation failed to increase p21 expression in BET, but was able to induce p53 and p21 in 59-2-HI and C7-2-HI tumors. The absence of an increased expression of p21 in E(2)-regressing BET lesions suggests that this protein is not necessary for estrogen-induced regression, but may be essential for antiprogestin action. Our results also suggest that p53/MDM2 alterations may be one of the mechanisms responsible for selected hormone resistance in breast carcinomas.