The genesis and unique properties of the lymphovascular tumor embolus are because of calpain-regulated proteolysis of E-cadherin.

The genesis and unique properties of the lymphovascular tumor embolus are poorly understood largely because of the absence of an experimental model that specifically reflects this important step of tumor progression. The lymphovascular tumor embolus is a blastocyst-like structure resistant to chemotherapy, efficient at metastasis and overexpressing E-cadherin (E-cad). Conventional dogma has regarded E-cad as a metastasis-suppressor gene involved in epithelial-mesenchymal transition. However, within the lymphovascular embolus, E-cad and its proteolytic processing by calpain and other proteases have a dominant oncogenic rather than suppressive role in metastasis formation and tumor cell survival. Studies using a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated the equivalence of xenograft-generated spheroids with lymphovascular emboli in vivo with both structures demonstrating E-cad overexpression and specific proteolytic processing. Western blot revealed full-length (FL) E-cad (120 kDa) and four fragments: E-cad/NTF1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa) and E-cad/NTF4 (80 kDa). Compared with MARY-X, only E-cad/NTF1 was present in the spheroids. E-cad/NTF1 was produced by calpain, E-cad/NTF2 by γ-secretase and E-cad/NTF3 by a matrix metalloproteinase (MMP). Spheroidgenesis and lymphovascular emboli formation are the direct result of calpain-mediated cleavage of E-cad and the generation of E-cad/NTF1 from membrane-associated E-cad rather than the de novo presence of either E-cad/NTF1 or E-cad/CTF1. E-cad/NTF1 retained the p120ctn-binding site but lost both the ß-catenin and α-binding sites, facilitating its disassembly from traditional cadherin-based adherens junctions and its 360° distribution around the embolus. This calpain-mediated proteolysis of E-cad generates the formation of the lymphovascular embolus and is responsible for its unique properties of increased homotypic adhesion, apoptosis resistance and budding.

A role for nucleotides in support of breast cancer angiogenesis: heterologous receptor signalling.

BACKGROUND: Human breast carcinoma cells secrete an adenosine 5'-diphosphate transphosphorylase (sNDPK) known to induce endothelial cell tubulogenesis in a P2Y receptor-dependent manner. We examined sNDPK secretion and its effects on human endothelial cells. METHODS: Nucleoside diphosphate kinase (NDPK) secretion was measured by western blot and enzyme-linked immunosorbent assay, while transphosphorylase activity was measured using the luciferin-luciferase ATP assay. Activation of MAPK was determined by western blot analysis, immunofluorescence and endothelial cell proliferation and migration. RESULTS: A panel of breast cancer cell lines with origin as ductal carcinoma, adenocarcinoma or medullary carcinoma, secrete sNDPK-A/B. Addition of purified NDPK-B to endothelial cultures activated VEGFR-2 and Erk(1/2), both of which were blocked by inhibitors of NDPK and P2Y receptors. Activation of VEGFR-2 and ErK(1/2) by 2-methylthio-ATP (2MeS-ATP) was blocked by pretreatment with the P2Y(1)-specific antagonist MRS2179, the proto-oncogene non-receptor tyrosine kinase (Src) inhibitor PP2 or the VEGFR-2 antagonist SU1498. Nucleoside diphosphate kinase-B stimulates cell growth and migration in a concentration-dependent manner comparable to the effect of vascular endothelial growth factor. Treatment of endothelial cells with either NDPK-B or 2MeS-ATP induced migration, blocked by P2Y(1), Src or VEGFR-2 antagonists. CONCLUSION: sNDPK supports angiogenesis. Understanding the mechanism of action of sNDPK and P2Y(1) nucleotide signalling in metastasis and angiogenesis represent new therapeutic targets for anti-angiogenic therapies to benefit patients.

The lymphovascular embolus of inflammatory breast cancer exhibits a Notch 3 addiction.

Inflammatory breast carcinoma (IBC) is characterized by exaggerated lymphovascular invasion (LVI), recapitulated in our human xenograft, MARY-X. This model exhibited lymphovascular emboli in vivo and corresponding spheroids in vitro. Owing to the morphological and gene profile resemblance of these spheroids to embryonal blastocysts, we wondered whether they might exhibit embryonic stem cell signaling. Specifically we investigated Notch and observed selective Notch 3 activation by expression profiling, reverse transcriptase- and real-time PCR, western blot and immunofluorescence in vitro, and immunohistochemistry in vivo. Notch 3 intracellular domain (N3icd) and six target genes, HES-5, HEY-1, c-Myc, Deltex-1, NRARP and PBX1, markedly increased in MARY-X. In addition, a significant percentage of MARY-X cells expressed aldehyde dehydrogenase (ALDH), a stem cell marker. Only the ALDH(+) cells were capable of secondary spheroidgenesis, tumorigenicity and self-renewal. Inhibiting Notch 3 activation in vitro with γ-secretase inhibitors (GSIs) or small interfering RNA resulted in a downregulation of Notch target genes, including CD133, and an induction of caspase 3-mediated apoptosis. Transfection of N3icd but not Notch 1 intracellular domain into normal human mammary epithelial cells resulted in increased expression of Notch target genes and induction of spheroidgenesis. GSI in vivo resulted in inhibitory but diffusion-limited effects on Notch 3 signaling, resulting in xenograft growth reduction. The lymphovascular emboli of human IBC exhibited dual N3icd and ALDH1 immunoreactivities independently of molecular subtype. This Notch 3 addiction of lymphovascular emboli might be exploited in future therapeutic strategies.

ERalpha signaling through slug regulates E-cadherin and EMT.

The ERalpha signaling pathway is one of the most important and most studied pathways in human breast cancer, yet numerous questions still exist such as how hormonally responsive cancers progress to a more aggressive and hormonally independent phenotype. We have noted that human breast cancers exhibit a strong direct correlation between ERalpha and E-cadherin expression by immunohistochemistry, suggesting that ERalpha signaling might regulate E-cadherin and implying that this regulation might influence epithelial-mesenchymal transition (EMT) and tumor progression. To investigate this hypothesis and the mechanisms behind it, we studied the effects of ERalpha signaling in ERalpha-transfected ERalpha-negative breast carcinoma cell lines, the MDA-MB-468 and the MDA-MB-231 and the effects of ERalpha knockdown in naturally expressing ERalpha-positive lines, MCF-7 and T47D. When ERalpha was overexpressed in the ERalpha-negative lines, 17beta-estradiol (E2) decreased slug and increased E-cadherin. Clones maximally exhibiting these changes grew more in clumps and became less invasive in Matrigel. When ERalpha was knocked down in the ERalpha-positive lines, slug increased, E-cadherin decreased, cells became spindly and exhibited increased Matrigel invasion. ERalpha signaling decreased slug expression by two different mechanisms: directly, by repression of slug transcription by the formation of a corepressor complex of ligand-activated ERalpha, HDAC inhibitor (HDAC1), and nuclear receptor corepressor (N-CoR) that bound the slug promoter in three half-site estrogen response elements (EREs); indirectly by phosphorylation and inactivation of GSK-3beta through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). The GSK-3beta inactivation, in turn, repressed slug expression and increased E-cadherin. In human breast cancer cases, there was a strong inverse correlation between slug and ERalpha and E-cadherin immunoreactivity. Our findings indicate that ERalpha signaling through slug regulates E-cadherin and EMT.

Semi-automated imaging system to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer.

A semi-automated imaging system is described to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer. The system works for any conventional method of image acquisition using microscopic slides that have been processed for immunohistochemical analysis of the estrogen receptor and progesterone receptor. Estrogen receptor and progesterone receptor immunohistochemical staining produce colorimetric differences in nuclear staining that conventionally have been interpreted manually by pathologists and expressed as percentage of positive tumoral nuclei. The estrogen receptor and progesterone receptor status of human breast cancer represent important prognostic and predictive markers of human breast cancer that dictate therapeutic decisions but their subjective interpretation result in interobserver, intraobserver and fatigue variability. Subjective measurements are traditionally limited to a determination of percentage of tumoral nuclei that show positive immunoreactivity. To address these limitations, imaging algorithms utilizing both colorimetric (RGB) as well as intensity (gray scale) determinations were used to analyze pixels of the acquired image. Image acquisition utilized either scanner or microscope with attached digital or analogue camera capable of producing images with a resolution of 20 pixels /10 mu. Areas of each image were screened and the area of interest richest in tumour cells manually selected for image processing. Images were processed initially by JPG conversion of SVS scanned virtual slides or direct JPG photomicrograph capture. Following image acquisition, images were screened for quality, enhanced and processed. The algorithm-based values for estrogen receptor and progesterone receptor percentage nuclear positivity both strongly correlated with the subjective measurements (intraclass correlation: 0.77; 95% confidence interval: 0.59, 0.95) yet exhibited no interobserver, intraobserver or fatigue variability. In addition the algorithms provided measurements of nuclear estrogen receptor and progesterone receptor staining intensity (mean, mode and median staining intensity of positive staining nuclei), parameters that subjective review could not assess. Other semi-automated image analysis systems have been used to measure estrogen receptor and progesterone receptor immunoreactivity but these either have required proprietary hardware or have been based on luminosity differences alone. By contrast our algorithms were independent of proprietary hardware and were based on not just luminosity and colour but also many other imaging features including epithelial pattern recognition and nuclear morphology. These features provide a more accurate, versatile and robust imaging analysis platform that can be fully automated in the near future. Because of all these properties, our semi-automated imaging system 'adds value' as a means of measuring these important nuclear biomarkers of human breast cancer.

Fiberoptic ductoscopy for breast cancer patients with nipple discharge.

BACKGROUND: Breast cancer and precancer are thought to originate in the lining of the milk duct, but until recently, we have not had direct access to this area other than in tissue removed blindly by core biopsy or fine-needle aspiration. Fiberoptic ductoscopy (FDS) is an emerging technique that allows direct visual access of the ductal system of the breast through nipple orifice cannulation and exploration. To date, this technique has been used only in pilot studies. Previously, we have demonstrated that fiberoptic ductoscopy in patients with and without nipple discharge is a safe and effective means of visualizing the intraductal lesion. When combined with cytology, it is a screening technique that has high predictive value. METHODS: We applied ductoscopy to 415 women with nipple discharge with the specific intent of detecting those patients with nipple discharge who had intraductal carcinoma (DCIS) as the basis of their discharge. RESULTS: In this cohort of patients, ductoscopy was successful in visualizing an intraductal lesion in 166 patients (40%). In these cases, ductal lavage following ductoscopy increased the yield of cytologically interpretable ductal epithelial cells 100-fold compared to discharge fluid alone. In the majority of these patients, FDS examination detected lesions that had the appearance of typical papillomas. However, in 10 patients, the intraductal lesion exhibited one of several atypical features, including bleeding, circumferential obstruction, and gross fungating projections. In eight of these patients, the subsequent histopathology turned out to be DCIS. In two of these eight patients, endoscopic biopsy revealed cytologically malignant cells; in two others, ductal lavage (washings) revealed cytologically malignant cells. In three additional patients, although FDS examination uncovered a typical papilloma that was not biopsied, ductal lavage (washings) revealed cytologically malignant cells. On surgical pathology review of the extirpated lesions, all 11 patients were subsequently shown to have DCIS. Of these 11 cases of DCIS that were initially detected with a combination of FDS and ductal lavage cytology, six were completely negative on mammogram and physical exam. CONCLUSION: Although nipple discharge is an unusual presentation for DCIS, in patients with nipple discharge, FDS with ductal lavage cytology is a useful technique for diagnosing DCIS prior to definitive surgery.

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.

Fiberoptic ductoscopy for patients with nipple discharge.

BACKGROUND: Breast carcinoma and precancer are thought to start in the lining of the milk duct or lobule, yet until recently, we have not had direct access to this area other than by blindly removing tissue by core biopsy or fine-needle aspiration. Fiberoptic ductoscopy (FDS) is an emerging technique allowing direct visual access to the ductal system of the breast through nipple orifice exploration. METHODS: We applied ductoscopy to 259 women who had nipple discharge, and we analyzed the visual findings, the cytological washings, and the subsequent histopathology. RESULTS: In 92 (36%) of these women, fiberoptic ductoscopy was successful in detecting an intraductal papillary lesion. Of these observed lesions, 68 (74%) were single papilloma, 21 (23%) were multiple discrete papillomas, and 3 (3%) were diffuse intraductal thickening which corresponded to diffuse papillomatosis on histopathological analysis. The overall positive predictive value of FDS screening was 83%. Of the lesions observed, 29.8% were located in the main (segmental) duct, 43.9% lesions in the first branch, 17.5% lesions in the second branch, 7.9% in the third branch, and 0.9% in the fourth branch. These lesions had an overall average distance of 2.7 cm from the nipple orifice. Ductal washings performed at the time of ductoscopy were effective at obtaining representative exfoliated ductal cells which could be evaluated for the presence of clumps (> 50 cells), clumps with atypia or single ductal cells. The presence of clumps with positive FDS increased the positive predictive value to 86%. CONCLUSIONS: Fiberoptic ductoscopy currently offers a safe alternative to ductography in guiding subsequent breast surgery in the treatment of nipple discharge.

Human breast carcinoma desmoplasia is PDGF initiated.

The desmoplastic response to human breast carcinoma is a host myofibroblast-mediated collagenous response exhibiting synergistic effects on tumor progression. Although many paracrine interactions between breast carcinoma cells and myofibroblasts have been characterized, the event(s) which initiate desmoplasia have remained undefined. Our studies utilized c-rasH transfected MCF-7 cells which overexpress ras p2l and which are weakly tumorigenic in ovariectomized nude mice. The xenografts are desmoplastic and comprised of 30% myofibroblasts and 60 mg/g of interstitial collagen. In situ hybridization studies of these xenografts reveal a stromal gene expression pattern (stromelysin-3, IGF-II and TIMP-1) identical to that observed in human tumor desmoplasia. 17-beta estradiol increases c-rasH MCF-7 growth but abolishes desmoplasia. c-rasH MCF-7 in vitro constitutively produce myofibroblast mitogenic activity which competes with PDGF in a receptor binding assay. This myofibroblast mitogenic activity is unaltered by 17-beta estradiol/tamoxifen pretreatment in vitro. Transfection of c-rasH MCF-7 with a PDGF-A dominant negative mutant, 1308, produced by site-directed mutagenesis (serine-->cysteine129) reduces both homo- and heterodimer secretion of PDGF by as much as 90% but does not interfere with the secretion of other growth factors. Clones with low PDGF, though tumorigenic, are non-desmoplastic. Our results suggest that breast carcinoma-secreted PDGF is the major initiator of tumor desmoplasia.

Evidence for a protein related immunologically to the jaagsiekte sheep retrovirus in some human lung tumours.

Human bronchioloalveolar carcinoma (BAC) is a lung cancer, morphologically similar to an endemic contagious lung neoplasm of sheep called sheep pulmonary adenomatosis (SPA) or jaagsiekte. SPA is caused by an exogenous type B/D retrovirus (jaagsiekte sheep retrovirus (JSRV)), which prompted the present study to obtain evidence of a retrovirus in BAC. A panel of 249 human lung tumours, 21 nontumour lung lesions, four normal lung tissues, 23 adenocarcinomas from other organs and a cell line expressing a human endogenous retrovirus protein was examined immunohistochemically using a rabbit antiserum directed against the JSRV capsid protein. Specific staining was detected only in the cytoplasm of recognizably neoplastic cells in the pulmonary alveoli of 39 of 129 (30%) BACs, 17 of 65 (26%) lung adenocarcinomas and two of seven large cell carcinomas. The remaining samples were negative. These results support the hypothesis that some human pulmonary tumours may be associated with a jaagsiekte sheep retrovirus-related retrovirus, warranting further studies.

Genistein's "ER-dependent and independent" actions are mediated through ER pathways in ER-positive breast carcinoma cell lines.

Genistein, a natural flavone found in soy has been postulated to be responsible for lowering the rate of breast cancer in Asian women. Our previous studies have shown that genistein exerts multiple suppressive effects on both estrogen receptor positive (ER+) as well as estrogen receptor negative (ER-) human breast carcinoma lines suggesting that the mechanisms of these effects may be independent of ER pathways. In the present study however we provide evidence that in the ER+ MCF-7, T47D and 549 lines but not in the ER-MDA-MB-231 and MDA-MB-468 lines both presumed "ER-dependent" and "ER-independent" actions of genistein are mediated through ER pathways. Genistein's antiproliferative effects are estrogen dependent in these ER+ lines, being more pronounced in estrogen-containing media and in the presence of exogenous 17-beta estradiol. Genistein also inhibits the expression of ER-downstream genes including pS2 and TGF-beta in these ER+ lines and this inhibition is also dependent on the presence of estrogen. Genistein inhibits estrogen-induced protein tyrosine kinase (PTK) activity. Genistein is only a weak transcriptional activator and actually decreases ERE-CAT levels induced by 17-beta estradiol in the ER+ lines. Genistein also decreases steady state ER mRNA only in the presence of estrogen in the ER+ lines thereby manifesting another suppression of and through the ER pathway. Our observations resurrect the hypothesis that genistein functions as a "good estrogen" in ER+ breast carcinomas. Since chemopreventive effects of genistein would be targeted to normal ER-positive ductal-lobular cells of the breast, this "good estrogen" action of genistein is most relevant to our understanding of chemoprevention.

Tamoxifen enhances myoepithelial cell suppression of human breast carcinoma progression in vitro by two different effector mechanisms.

Our previous studies have indicated that myoepithelial cells surrounding ductal and acinar epithelium of glandular organs, such as the breast, exert multiple paracrine suppressive effects on incipient and developing cancers that arise from this epithelium. Myoepithelial cells and derived cell lines (HMS 1-6) exert these effects through the secretion of a number of different effector molecules that exert anti-invasive, anti-proliferative, and anti-angiogenic activities. Since previous basic and clinical studies have examined the role of estrogen agonists and antagonists on human breast cancer cells and because issues of hormone replacement therapy (HRT) and tamoxifen chemoprevention are such timely issues in breast cancer, we wondered whether or not hormonal manipulations might affect myoepithelial cells in vitro as far as their paracrine suppressive activities on breast cancer were concerned. The present in vitro study demonstrates that treatment of myoepithelial cells with tamoxifen but not 17beta-estradiol increases both maspin secretion and invasion-blocking ability. Furthermore tamoxifen but not 17beta-estradiol increases inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by myoepithelial cells when they are co-cultured with conditioned media from or breast carcinoma cells directly. This increased myoepithelial NO exerts both autocrine and paracrine antiproliferative effects which can be blocked by inhibition of iNOS. 17beta-Estradiol, however, competes with all of these suppressive effects of tamoxifen suggesting that the mechanism of tamoxifen action is estrogen receptor mediated. Myoepithelial cells lack ER-alpha but express ER-beta. Tamoxifen, but not 17beta-estradiol, increases AP-1 CAT but not ERE-CAT activity. Again, 17beta-estradiol competes with the transcription-activating effects of tamoxifen. These experiments collectively suggest that the actions of tamoxifen on the increased secretion of maspin and increased production of NO by myoepithelial cells are mediated through ER-beta and the transcription-activation of an ER-dependent AP-1 response element.

Breast-cancer diagnosis with nipple fluid bFGF.

Early diagnosis of breast cancer is the key to extending survival of breast-cancer patients. We found that the concentrations of nipple fluid bFGF (basic fibroblast growth factor) was significantly increased in breast-cancer patients compared with concentrations in controls (1717 ng/L [SD 706] vs 19 ng/L [19]; Student's t test p=0.027). Measurement of bFGF in nipple fluid could be a useful diagnostic tool for breast cancer, and deserves further study.

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

Uterine smooth muscle tumors: utility of classification by proliferation, ploidy, and prognostic markers versus traditional histopathology.

CONTEXT: Accurate categorization of uterine smooth muscle neoplasms by light microscopic examination is difficult. Multiple classification schemes have been proposed based on mitotic rate, nuclear atypia, and the presence or absence of necrosis. None of these classification systems has been entirely successful. Multiple ancillary techniques have been tested for their ability to predict behavior of uterine smooth muscle tumors. OBJECTIVE: We assayed 45 smooth muscle neoplasms for a variety of proliferation markers, oncogene protein products, and DNA ploidy level to determine if these markers supplied prognostically useful information over and above that obtained by routine light microscopic assessment. STUDY DESIGN: Forty-five uterine smooth muscle neoplasms were assessed for DNA ploidy; silver-staining nucleolar organizer regions (AgNORs); percent nuclear proliferating cell nuclear antigen (PCNA); expression of p53, Her-2/neu, and MDM-2 protein; mitotic rate; and nuclear grade. These markers were correlated with histologic diagnosis and the occurrence of a clinically adverse event (death, metastasis, or recurrence). RESULTS: Diagnostic category (P <.001), nuclear grade (P <.002), mitotic activity (P <.001), mean AgNORs (P <.001), percent nuclear PCNA (P =.02), and expression of p53 (P =.02) all correlated with clinical outcome. No statistically significant correlation between clinical outcome and the categories MDM-2 expression, Her-2/neu expression, or DNA ploidy was seen. Nuclear grade, p53 expression, mitotic rate, AgNORs, and percent nuclear PCNA correlated with diagnosis. CONCLUSIONS: Diagnostic category, mitotic rate, AgNOR counts, PCNA, and p53 expression dichotomized uterine smooth muscle neoplasms into prognostically favorable and unfavorable groups. Although highly significant, the category AgNORs was no more successful than mitotic rate in dividing uterine smooth muscle neoplasms into prognostically favorable and unfavorable groups. Expression of p53 and percent nuclear PCNA dichotomized uterine smooth muscle neoplasms into prognostic groups, but neither technique reached the level of significance achieved by mitotic rate. Our data indicate that mitotic rate and the classification system of Kempson and Bari are at least as effective as the tested markers in separating uterine smooth muscle neoplasms into prognostic categories.

Human colon cancer cells express multiple glycoprotein ligands for E-selectin.

The interaction between the colon tumor cell surface and the endothelial cell layer is an important component of tumor intravasation, extravasation, and metastasis. Multiple studies suggest that tumor cells may bind to E-selectin expressed on endothelial cells during these processes. To identify possible E-selectin ligands on tumor cells that may participate in this mechanism, we used E-selectin-Ig chimera affinity chromatography to isolate glycoproteins from the human colon cancer cell line Colo-205. Binding of these cells to E-selectin was specific, required the presence of calcium, and could be blocked by antibodies against E-selectin. We identified LAMP-1 (lysosomal membrane glycoprotein-1), LAMP-2, and two high molecular weight glycoproteins (>400 kDa and 300 kDa) as the main E-selectin ligands on Colo-205 cells. Treatment of the cells with N-glycanase and O-sialoglycoprotease abolished their binding to E-selectin. The high MW glycoproteins contained sialyl Lewis X and/or sialyl Lewis A glycoconjugates, and appeared to be either alternatively spliced or alternatively glycosylated forms of MUC-1 (mucin-1).

Different patterns of angiogenesis in sarcomas and carcinomas.

Solid tumors depend on angiogenesis for growth and metastasis. It has been shown that blood vessel density, as determined by counting the number of capillaries in clustered bursts, is a significant prognostic factor in carcinomas. It is unclear, however, whether vessel density is a prognostic factor in sarcomas. In this study, we examined angiogenesis in sarcomas of various grades and compared their vascular patterns to those of carcinomas. Microvessels were identified by von Willebrand factor staining. The matrix of multiple sarcoma and breast carcinoma specimens were extracted and subjected to Western analysis of various angiogenic factors and inhibitors. Metalloproteinase inhibitor presence was also determined by in situ hybridization. In breast carcinomas, capillaries were clustered in bursts within the stroma of the tumor, whereas the sarcoma capillaries were homogeneously distributed in the tumor stroma. Random blood vessel density per high power field in sarcomas did not correlate with patient prognosis. The matrix of sarcomas and carcinomas contained both angiogenic stimulators and inhibitors. Tissue inhibitor of metalloproteinase-1 was found predominantly in fibroblasts and myofibroblasts in the matrix of carcinoma specimens. The difference in the pattern of angiogenesis in sarcomas and carcinomas may be attributable to the presence of fibroblasts and myofibroblasts in carcinomas, resulting in the compartmentalization of bursts of angiogenic factors. The homogeneous appearance of vessel density in sarcomas observed in the present study would be the consequence of the influence of a single compartment.

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.