Hormone Rhythms and Breast Cancer Chronoepidemiology: Salivary Progesterone Concentrations in Pre- and Post-Menarchal Girls and in Normal Premenopausal Women

Orcadian and circatrigintan time series of salivary progesterone levels in premenarchal and adolescent girls and healthy mature premenopausal women have been investigated as a possible determinant for breast cancer risk. Circadian variations in progesterone appear to be more random than systematic and estimates of total daily progesterone output are better represented by samples pooled from several 2-hr specimens. Different patterns of circatrigintan progesterone secretion in girls are recognised and relate to those experienced in infertile and fertile women, though their relation to chronological or menarchal age is as yet uncertain. These data suggest that the measurement of salivary progesterone at premenarche, adolescence and maturity is a feasible, though statistically difficult, study for prospective identification of individuals at risk for breast cancer.     

The transient and constitutive inflammatory signaling in tumorigenesis

Comment on: Rokavec M, et al. Mol Cell 2012; 45:777-89.     

Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

Estrogen-induced lysosomal proteases secreted by breast cancer cells: A role in carcinogenesis?

In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75-1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin-D-like aspartyl protease bearing mannose-6-phosphate signals. This precursor displays an in vitro autocrine mitogenic activity on estrogen-deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin-D-like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin-B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin-L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.     

Evaluation of fine needle aspiration of the male breast for the diagnosis of gynaecomastia

Fine needle aspiration of the male breast can present problems of diagnosis because the cytological presentation of gynaecomastia can be confused with that of adenocarcinoma. We reviewed breast aspirates from 24 male patients in order to determine the accuracy of cytology as a method of diagnosing gynaecomastia. Discrepancies were observed between the original cytology reports on one hand and the review cytology and biopsies on the other. Of the 24 aspirates from the male breast, the cytology was reported as negative in 16 cases, suspicious in three cases and malignant in five. In four cases of the negative group, a specific diagnosis of gynaecomastia was made. In two of the negative cases the subsequent biopsies revealed adenocarcinoma. Of the five cases reported on the original cytology as adenocarcinoma, two on review showed the features of florid gynaecomastia and this was confirmed on biopsy and three confirmed the initial diagnosis of adenocarcinoma. The cytological features of gynaecomastia which distinguish it from adenocarcinoma are discussed.     

Cytogenetic diversity in primary human tumors

Cytogenetic patterns from primary short-term culture of breast cancer, renal carcinoma, and tumors of the central nervous system are presented to illustrate the range of karyotypic diversity of human solid tumors as well as their biologic differences in culture systems that support their growth. These studies have illustrated several major issues. 1) Results vary with the tissue of origin: primary cultures from breast are almost uniformly diploid, while renal tumors are near-diploid, mosaic, and show clonal aberrations; and CNS tumors are heterogeneous: some diploid, some near-diploid and some highly aneuploid. 2) Results after short-term culture are selective, representing subpopulations from the heterogeneous cells that are detected on direct analysis of fresh tumors by cytogenetics or flow cytometry (FCM). It is not yet clear whether prognosis depends on the dominant population of the primary tumor or alternatively should be influenced by detection of small aneuploid subpopulations. 3) Evidence from all three tumor types supports the interpretation that cytogenetically normal diploid cells constitute part of some tumor populations, and may be better adapted to routine growth in culture than aneuploid subpopulations from the same primary tumors. These cells may also compose a major portion of the viable population of tumors in vivo and, therefore, could represent a useful model for studies of tumorigenesis and therapeutic regimens.     

Oncogene expression in mammary epithelial cells.

Mouse strains which develop tumors at a high incidence with characteristics very similar to human cancers have been derived over the last 8 years. The tumors are caused by defined genetic alterations in the mouse genome. Three areas of research have contributed to the derivation of these mouse strains: (1) Molecular analysis of human tumors has shown that distinct oncogenes and tumor suppressor genes are consistently involved in a high percentage of primary tumors. (2) Regulatory enhancer-promoter sequences have been identified which direct gene expression to specific target cells, preferentially mammary epithelial cells. (3) The introduction of recombinant DNA molecules into fertilized mouse eggs by microinjection and integration of the injected DNA into the genome of injected cells has given rise to mutant mouse strains with unique and defined genetic alterations. Studies with different promoter-oncogene combinations introduced into transgenic mouse strains have led to the following general conclusions: (1) Oncogenes expressed in mammary gland cells predispose transgenic mice to mammary tumors. (2) The oncogenic potential of individual oncogenes in mammary epithelial cells differs. (3) Oncogene expression initially often causes a preneoplastic state affecting growth and differentiation parameters of cells. (4) The expression of different oncogenes synergizes to reduce tumor latency. Synergism can also be observed with physiological growth signals like estrogen or growth hormone. The oncogenes with a role in mammary carcinomas which have been investigated in transgenic mice will be described here. The phenotypic consequences of oncogene expression and the implications for the multistep carcinogenesis model will be discussed.     

miR-145通过靶向吞噬和细胞活力蛋白1抑制乳腺癌细胞侵袭

吞噬和细胞活力蛋白1（engulfment and cell motility protein 1,ELMO1）可以促进多种癌细胞的侵袭和转移,但ELMO1的表达是否受miRNA的调控鲜有研究。本研究旨在探讨miR-145与ELMO1表达的相关性,以及miR-145通过结合ELMO1的mRNA对乳腺癌侵袭的影响。通过TargetScan (http://www.targetscan.org/)靶基因预测软件预测与ELMO1的3′UTR结合的miR-145。荧光素酶结果证实两者互补结合。Transwell侵袭结果显示,miR-145组和siELMO1+miR-145组MDA-231乳腺癌细胞穿膜数较对照组分别降低40%（P＜0.05）和79%（P＜0.05）。siELMO1+miR-145组和siELMO1组细胞穿膜数则无显著差异（P>0.05）。结果提示,miR-145通过与ELMO1的mRNA结合抑制细胞侵袭。qRT-PCR显示,低侵袭的MCF-7乳腺癌细胞miR-145的表达量较高侵袭的MDA-435细胞高80%（P＜0.05）,较MDA-231乳腺癌细胞高75%（P＜0.05）,即miR-145与癌细胞侵袭能力呈负相关。Western印迹结果表明,miR-145组ELMO1表达量低于阴性对照组,miR-145 抑制组ELMO1表达量高于抑制剂NC组（P＜0.05）,证明miR-145抑制ELMO1的表达。qRT-PCR显示,过表达miR-145后ELMO1 mRNA含量与对照组无显著差异（P>0.05）。结果提示,miR-145对ELMO1的调控作用通过抑制其翻译实现。F-肌动蛋白聚合实验表明,miR-145组和阴性对照组于20 s和60 s时F-肌动蛋白聚合结果存在明显区别（P＜0.05）。Western 印迹结果表明,miR-145组活化的Rac1表达量较阴性对照组降低60%（P<0.05）,抑制剂NC组活化的Rac1较miR-145 抑制组降低55%（P<0.05）;miR-145组磷酸化的整合素β1较对照组于15 min时降低42%（P<0.05）,于30 min时降低31%（P<0.05）。由此得出的miR-145过表达显著促进乳腺癌细胞F-肌动蛋白聚合、Rac1活化和整合素β1磷酸化结论。综上所述,miR-145通过靶向ELMO1的 mRNA抑制ELMO1翻译,从而抑制乳腺癌的侵袭。