G. Shyamala

Pathway Pathology: Histological Differences Between ErbB/Ras and Wnt Pathway Transgenic Mammary Tumors

To study phenotype-genotype correlations, ErbB/Ras pathway tumors (transgenic for ErbB2, c-Neu, mutants of c-Neu, polyomavirus middle T antigene (PyV-mT), Ras, and bi-transgenic for ErbB2/Neu with ErbB3 and with progesterone receptor) from four different institutions were histopathologically compared with Wnt pathway tumors [transgenes Wnt1, Wnt10b, dominant-negative glycogen synthase kinase 3-beta, beta-Catenin, and spontaneous mutants of adenomatous polyposis coli gene (Apc)]. ErbB/Ras pathway tumors tend to form solid nodules consisting of poorly differentiated cells with abundant cytoplasm. ErbB/Ras pathway tumors also have scanty stroma and lack myoepithelial or squamous differentiation. In contrast, Wnt pathway tumors exhibit myoepithelial, acinar, or glandular differentiation, and, frequently, combinations of these. Squamous metaplasia is frequent and may include transdifferentiation to epidermal and pilar structures. Most Wnt pathway tumors form caricatures of elongated, branched ductules, and have well-developed stroma, inflammatory infiltrates, and pushing margins. Tumors transgenic for interacting genes such as protein kinase CK2alpha (casein kinase IIalpha), and the fibroblast growth factors (Fgf) Int2/Fgf3 or keratinocyte growth factor (Kgf/Fgf7) also have the Wnt pathway phenotype. Because the tumors from the ErbB/Ras and the Wnt pathway are so distinct and can be readily identified using routine hematoxylin and eosin sections, we suggest that pathway pathology is applicable in both basic and clinical cancer research.

Latent transforming growth factor-β activation in mammary gland - Regulation by ovarian hormones affects ductal and alveolar proliferation

Transforming growth factor-beta1 (TGF-beta 1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-beta 1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-beta. Activation was highly restricted. TGF-beta 1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-beta 1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-beta 1 immunoreactivity, which suggests that TGF-beta 1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-beta 1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-beta 1 activity, we manipulated TGF-beta 1 levels in vivo using Tgfbeta 1 knockout mice and undertook tissue recombination experiments with heterozygous tissue. In Tgfbeta 1 heterozygous mice, which have <10% wild-type levels of TGF-beta1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgfbeta 1+/- epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-beta1 was critical to the proliferative phenotype, Tgfbeta 1+/+ and +/- epithelium were transplanted into +/+ mammary stroma. The outgrowth of Tgfbeta 1+/- epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgfbeta 1+/- than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-beta 1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-beta 1 activation, which in turn restricts proliferative response to hormone signaling.

Proliferation of estrogen receptor-α-positive mammary epithelial cells is restrained by transforming growth factor-β1 in adult mice

Transforming growth factor (TGF)-β1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-α cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-β1 is necessary for the quiescence of ER-α-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-β1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF-β signaling is autocrine. Nuclear Smad co-localized with nuclear ER-α. To test whether TGF-β inhibits proliferation, we examined genetically engineered mice with different levels of TGF-β1. ER-α co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgfβ1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-β1 via the MMTV promoter suppressed proliferation of ER-α-positive cells. Thus, TGF-β1 activation functionally restrains ER-α-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-β1 dysregulation may promote proliferation of ER-α-positive cells associated with breast cancer risk in humans.

Id-1 is not expressed in the luminal epithelial cells of mammary glands

BackgroundThe family of inhibitor of differentiation/DNA binding (Id) proteins is known to regulate development in several tissues. One member of this gene family, Id-1, has been implicated in mammary development and carcinogenesis. Mammary glands contain various cell types, among which the luminal epithelial cells are primarily targeted for proliferation, differentiation and carcinogenesis. Therefore, to assess the precise significance of Id-1 in mammary biology and carcinogenesis, we examined its cellular localization in vivo using immunohistochemistry.MethodsExtracts of whole mammary glands from wild type and Id-1 null mutant mice, and tissue sections from paraffin-embedded mouse mammary glands from various developmental stages and normal human breast were subjected to immunoblot and immunohistochemical analyses, respectively. In both these procedures, an anti-Id-1 rabbit polyclonal antibody was used for detection of Id-1.ResultsIn immunoblot analyses, using whole mammary gland extracts, Id-1 was detected. In immunohistochemical analyses, however, Id-1 was not detected in the luminal epithelial cells of mammary glands during any stage of development, but it was detected in vascular endothelial cells.ConclusionId-1 is not expressed in the luminal epithelial cells of mammary glands.

Effect of c-neu/ ErbB2 Expression Levels on Estrogen Receptor α–Dependent Proliferation in Mammary Epithelial Cells: Implications for Breast Cancer Biology

Mammary development and tumorigenesis are profoundly influenced by signaling pathways under the control of c-erbB2/c-neu and estrogen receptor alpha (ERalpha). Signaling through ERalpha is essential for ductal growth during puberty. In mice overexpressing wild-type c-neu in mammary epithelial cells, Tg (c-neu), ductal growth is impaired. An impeded signaling through ERalpha is also observed in a subset of human mammary tumors that overexpress erbB2. However, ductal growth is also impaired in the absence of c-neu in mouse mammary epithelial cells. To resolve this apparent paradox, we examined the relationship between c-neu expression and estrogen/ERalpha-dependent cell proliferation in pubertal Tg (c-neu). We report that proliferation in both terminal end buds and ducts is associated with ERalpha-positive cells, including those that coexpress c-neu, and is abolished in the absence of circulating estradiol. Tg (c-neu) contains hyperplastic mammary ducts with high proliferative index and coexpression of both ERalpha and c-neu in the dividing cells. These findings suggest that c-neu promotes ERalpha-dependent proliferation, and that this is responsible for the presence of hyperplastic ducts. Some of the hyperplastic ducts have acinar structures, indicative of morphologic differentiation. These ducts have low proliferative index and accompanied by a vast decrease in proliferation of ERalpha-positive cells, including those that express c-neu. As such, c-neu has dual but opposing effects on ERalpha-dependent proliferation in mammary epithelial cells. Therefore, depending on the physiologic setting, ductal morphogenesis will be compromised both in the absence and overexpression of c-neu, thus explaining the paradox.

Overexpression of progesterone receptor A isoform in mice leads to endometrial hyperproliferation, hyperplasia and atypia

A delicate balance in estrogen and progesterone signaling through their cognate receptors is characteristic for the physiologic state of the endometrium, and a shift in receptor isotype expression can be frequently found in human endometrial pathology. In this study, using a transgenic mouse model, we examined the mechanisms whereby alterations in progesterone receptor (PR) isotype expression leads to endometrial pathology. For an experimental model, we used transgenic mice (PR-A transgenics) carrying an imbalance in the native ratio of the two PR isoforms A and B (PR-A and PR-B) through the expression of additional A form and examined their uterine phenotype under different hormonal regimens, using various criteria. Uterine epithelial cell proliferation was augmented in PR-A transgenics and was abolished by PR antagonists. In particular, proliferative response to progesterone, independent of signaling through estrogen, was enhanced. Upon continuous exposure to estradiol and progesterone, the uteri in PR-A transgenics displayed gross enlargement, endometrial hyperplasia including atypical lesions, endometritis and pelvic inflammatory disease. Imbalanced expression of the two isoforms of PR in a transgenic model reveals multiple derangements in the regulation of uterine physiology, resulting in various pathologies including hyperplasias.