An intact overexpressed E-cadherin/alpha,beta-catenin axis characterizes the lymphovascular emboli of inflammatory breast carcinoma.

The step of intravasation (lymphovascular invasion), a rate-limiting step in metastasis, is greatly exaggerated in inflammatory breast carcinoma (IBC). Because nearly all human breast carcinoma cell lines grow as solitary nodules in nude/severe combined immunodeficient mice without manifesting lymphovascular invasion, this step has been difficult to study. We captured the essence of the IBC phenotype by establishing a unique human transplantable IBC xenograft, MARY-X, which manifests florid lymphovascular emboli in severe combined immunodeficient/nude mice. Comparing MARY-X with common non-IBC cell lines/xenografts, we discovered an overexpressed and overfunctioning E-cadherin/alpha,beta-catenin axis. In MARY-X, the E-cadherin and catenins were part of a structurally and functionally intact adhesion axis involving the actin cytoskeleton. In vitro, MARY-X grew as round compact spheroids with a cell density 5-10-fold higher than that of other lines. The spheroids of MARY-X completely disadhered when placed in media containing absent Ca(2+) or anti-E-cadherin antibodies or when retrovirally transfected with a dominant-negative E-cadherin mutant (H-2K(d)-E-cad). Anti-E-cadherin antibodies injected i.v. immunolocalized to the pulmonary lymphovascular emboli of MARY-X and caused their dissolution. H-2K(d)-E-cad-transfected MARY-X spheroids were only weakly tumorigenic and did not form lymphovascular emboli. A total of 90% of human IBCs showed increased membrane E-cadherin/alpha,beta-catenin immunoreactivity. These findings indicate that it is the gain and not the loss of the E-cadherin axis that contributes to the IBC phenotype.

Myoepithelial-specific CD44 shedding is mediated by a putative chymotrypsin-like sheddase.

Our previous studies have demonstrated that myoepithelial cells, which surround incipient carcinomas in situ of the breast and other organs, exert antiinvasive and antiangiogenic effects in vitro through the elaboration of a number of different suppressor molecules among which include the shed membrane CD44. The present study addresses the mechanism of this myoepithelial CD44 shedding. This CD44 shedding is enhanced by PMA pretreatment, is specific for myoepithelial CD44, and inhibited by chymotrypsin-like inhibitors (chymostatin, alpha(1)-antichymotrypsin, TPCK, and SCCA-2) but not by trypsin-like inhibitors (TLCK), nor papain-like inhibitors (SCCA-1) nor hydroxamate-based or general metalloproteinase inhibitors (BB2516 (marimastat), 1,10-phenanthroline, and TIMP-1). The effect of PMA can be mimicked by exogenous chymotrypsin but not by other proteases. The CD44 shedding activity cannot be transferred by conditioned media, cell-cell contact, peripheral membrane, or integral membrane fractions. However, cell-free purified integral plasma membrane fractions obtained from myoepithelial cells pretreated with PMA also exhibit CD44 shedding which is inhibited by chymotrypsin-like inhibitors. These findings support the presence and activation of a putative chymotrypsin-like sheddase as the mechanism of CD44 shedding in myoepithelial cells.

Myoepithelial-specific CD44 shedding contributes to the anti-invasive and antiangiogenic phenotype of myoepithelial cells.

Myoepithelial cells surround incipient ductal carcinomas of the breast and exert anti-invasive and antiangiogenic effects in vitro through the elaboration of suppressor molecules. This study examines one putative molecule, solubilized CD44 produced by myoepithelial shedding of membrane CD44. Studies with different human myoepithelial cell lines demonstrate that myoepithelial cells express and shed both the 85-kDa standard (CD44s) and the 130-kDa epithelial (CD44v8-10) isoforms, findings further confirmed by the use of isoform-specific antibodies. PMA pretreatment enhances CD44 shedding detected by two different methods at different time points: a reduction in surface CD44 at 2 h by flow cytometry and a marked decrease in both total cellular CD44 and plasma membrane CD44 at 12 h by Western blot. This shedding is both specific for CD44 and specific for myoepithelial cells. This shedding is inhibited by the chymotrypsin inhibitors chymostatin and alpha(1)-antichymotrypsin but not by general metallo-, cysteine, or other serine proteinase inhibitors. Myoepithelial-cell-conditioned medium and affinity-purified solubilized CD44 from this conditioned medium block hyaluronan adhesion and migration of both human carcinoma cell lines and human umbilical vein endothelial cells.

The human myoepithelial cell displays a multifaceted anti-angiogenic phenotype.

Human myoepithelial cells which surround ducts and acini of certain organs such as the breast form a natural border separating epithelial cells from stromal angiogenesis. Myoepithelial cell lines (HMS-1-6), derived from diverse benign myoepithelial tumors, all constitutively express high levels of active angiogenic inhibitors which include TIMP-1, thrombospondin-1 and soluble bFGF receptors but very low levels of angiogenic factors. These myoepithelial cell lines inhibit endothelial cell chemotaxis and proliferation. These myoepithelial cell lines sense hypoxia, respond to low O2 tension by increased HIF-1 alpha but with only a minimal increase in VEGF and iNOS steady state mRNA levels. Their corresponding xenografts (HMS-X-6X) grow very slowly compared to their non-myoepithelial carcinomatous counterparts and accumulate an abundant extracellular matrix devoid of angiogenesis but containing bound angiogenic inhibitors. These myoepithelial xenografts exhibit only minimal hypoxia but extensive necrosis in comparison to their non-myoepithelial xenograft counterparts. These former xenografts inhibit local and systemic tumor-induced angiogenesis and metastasis presumably from their matrix-bound and released circulating angiogenic inhibitors. These observations collectively support the hypothesis that the human myoepithelial cell (even when transformed) is a natural suppressor of angiogenesis. Oncogene (2000) 19, 3449 - 3459

A novel human xenograft model of inflammatory breast cancer.

The step of intravasation or lymphovascular invasion can be a rate-limiting step in the metastatic process. Inflammatory breast carcinoma manifests an exaggerated degree of lymphovascular invasion in situ; hence, a study of its molecular basis might shed light on the general mechanism of lymphovascular invasion exhibited by all metastasizing cancers. To this end, we have established the first human transplantable inflammatory breast carcinoma xenograft (MARY-X) in scid/nude mice. Whereas all other human xenografts grew as isolated s.c. nodules, MARY-X grew exclusively within murine lymphatics and blood vessels, and these latter elements and their supporting stroma comprised, by murine Cot-1 DNA analysis, 30% of the tumor. MARY-X, like its human counterpart, exhibited striking erythema of the overlying skin. MARY-X was estrogen receptor, progesterone receptor, Her-2/neu negative and p53, epidermal growth factor receptor positive. The primary tumor of origin of MARY-X exhibited identical markers, except that about 50% of its cells exhibited Her-2/neu amplification. Comparative studies of MARY-X with noninflammatory xenografts indicated 10-20-fold overexpression of E-cadherin and MUC1, findings that were reflected in actual cases of human inflammatory breast cancer. MARY-X should allow us to further dissect out both the upstream regulatory machinery and the downstream effector molecules responsible for the inflammatory carcinoma phenotype.

The human myoepithelial cell exerts antiproliferative effects on breast carcinoma cells characterized by p21WAF1/CIP1 induction, G2/M arrest, and apoptosis.

Ductal carcinoma in situ of the breast (DCIS) is surrounded by a layer of myoepithelial cells. Our previous studies have suggested that these myoepithelial cells exert paracrine tumor-suppressive effects on invasion of breast carcinoma cells. Conditioned medium (CM), concentrated 10-100x of HMS-1, HMS-3, and HMS-4, human myoepithelial cell lines, block Matrigel invasion of a series of carcinoma cell lines. Immunoprecipitation of maspin, a recently described serpin, from these CM abolishes this anti-invasive effect. Both CM and maspin-immunoprecipitated CM, however, exert equal antiproliferative effects on a series of ER+ and ER- cell lines including MCF-7, T47D, MDA-MB-231, and MDA-MB-468. These antiproliferative effects are characterized by induction of a G2/M arrest, a twofold increase in p21(WAF1/CIP1) transcription and expression, and a threefold increase in apoptosis in the breast carcinoma lines examined. The antiproliferative effects mediated by myoepithelial cell CM do not manifest themselves in an autocrine manner, are not mediated by TGF-beta1, nor involve ER- or p53-dependent pathways. Neither the antiproliferative nor the anti-invasive effects of myoepithelial cell CM is observed with nonmyoepithelial cell CM. The in vitro observations of our present study may have relevance in explaining the increased degree of apoptosis exhibited by DCIS cells in vivo. Our findings illustrate another way myoepithelial cells function as natural paracrine tumor suppressors.

Genistein inhibits proliferation similarly in estrogen receptor-positive and negative human breast carcinoma cell lines characterized by P21WAF1/CIP1 induction, G2/M arrest, and apoptosis.

Genistein has been proposed to be responsible for lowering the rate of breast cancer in Asian women but the mechanism for this chemopreventive effect in vivo is unknown. In this study, we present in vitro evidence that genistein inhibits cell proliferation similarly in ER-positive and ER-negative human breast carcinoma cell lines. This inhibition is associated with specific G2/M arrest and induction of p21WAF1/CIP1 expression. Genistein results in a five-to six-fold increase in p21WAF1/CIP1 mRNA levels and a three- to four-fold increase in protein levels, only a 1.5-fold increase in p21WAF1/CIP1 transcription but a three- to six-fold increase in p21WAF1/CIP1 mRNA stability. The increase in p21WAF1/CIP1 is followed by increased apoptosis. The similar effects of genistein on a number of breast carcinoma cell lines with different ER and p53 status suggest that the actions of genistein reported here are mediated through ER and p53 independent mechanisms. The chemopreventive effects of genistein in vivo could be mediated along an identical or similar anti-proliferative pathway.