Cases Of Human Breast Carcinoma Research Articles

Krüppel-like factor 5 in human breast carcinoma: a potent prognostic factor induced by androgens

Krüppel-like factor 5 (intestinal) or Krüppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor and involved in important biological processes including cell proliferation and differentiation. However, clinical significance of KLF5 protein has remained largely unknown in breast cancer. Therefore, in this study, we immunolocalized KLF5 in 113 human breast carcinoma cases. KLF5 immunoreactivity was frequently detected in the nuclei of breast carcinoma cells, and median value of the ratio of KLF5-positive carcinoma cells was 30% and was positively associated with the status of androgen receptor. KLF5 immunoreactivity was also significantly associated with increased risk of recurrence and worse clinical outcome in breast cancer patients by univariate analyses, and subsequent multivariate analyses demonstrated that KLF5 immunoreactivity was an independent prognostic factor for both disease-free and breast cancer-specific survival of the patients. We then examined possible regulation of KLF5 by androgen using MCF-7 breast carcinoma cells. KLF5 mRNA was induced by biologically active androgen 5α-dihydrotestosterone in a dose- and time-dependent manner in MCF-7 cells. In addition, results of transfection experiments demonstrated that proliferation activity of MCF-7 cells was significantly associated with the KLF5 expression level. These findings suggest that KLF5 is an androgen-responsive gene in human breast carcinomas and play important roles in the progression of breast carcinomas. KLF5 immunoreactivity is therefore considered a potent prognostic factor in human breast cancers.

Old proteins – new locations: myoglobin, haemoglobin, neuroglobin and cytoglobin in solid tumours and cancer cells

The unexpected identification of myoglobin (MB) in breast cancer prompted us to evaluate the clinico-pathological value of MB, haemoglobin (HB) and cytoglobin (CYGB) in human breast carcinoma cases. We further screened for the presence of neuroglobin (NGB) and CYGB in tumours of diverse origin, and assessed the O(2) -response of HB, MB and CYGB mRNAs in cancer cell lines, to better elicit the links between this ectopic globin expression and tumour hypoxia. Breast tumours were analysed by immunohistochemistry for HB, MB and CYGB and correlated with clinico-pathological parameters. Screening for CYGB and NGB mRNA expression in tumour entities was performed by hybridization, quantitative PCR (qPCR) and bioinformatics. Hypoxic or anoxic responses of HB, MB and CYGB mRNAs was analysed by qPCR in human Hep3B, MCF7, HeLa and RCC4 cancer cell lines. 78.8% of breast cancer cases were positive for MB, 77.9% were positive for HB and 55.4% expressed CYGB. The closest correlation with markers of hypoxia was observed for CYGB. Compared to the weakly positive status of MB in healthy breast tissues, invasive tumours either lost or up-regulated MB. Breast carcinomas showed the tendency to silence CYGB. HB was not seen in normal tissues and up-regulated in tumours. Beyond breast malignancies, expression levels of NGB and CYGB mRNAs were extremely low in brain tumours (glioblastoma, astrocytoma). NGB was not observed in non-brain tumours. CYGB mRNA, readily detectable in breast cancer and other tumours, is down-regulated in lung adenocarcinomas. Alpha1 globin (α1 globin) and Mb were co-expressed in MCF7 and HeLa cells; CYGB transcription was anoxia-inducible in Hep3B and RCC4 cells. This is the first time that HB and CYGB are reported in breast cancer. Neither NGB nor CYGB are systematically up-regulated in tumours. The down-regulated CYGB expression in breast and lung tumours is in line with a tumour-suppressor role. Each of the screened cancer cells expresses at least one globin (i.e. main globin species: CYGB in Hep3B; α1 globin + MB in MCF7 and HeLa). Thus, globins exist in a wide variety of solid tumours. However, the generally weak expression of the endogenous proteins in the cancer argues against a significant contribution to tumour oxygenation. Future studies should consider that cancer-expressed globins might function in ways not directly linked to the binding and transport of oxygen.

VRK2 Inhibits Mitogen-Activated Protein Kinase Signaling and Inversely Correlates with ErbB2 in Human Breast Cancer

The epidermal growth factor (EGF)-ErbB-mitogen-activated protein kinase (MAPK) transcription signaling pathway is altered in many types of carcinomas, and this pathway can be regulated by new protein-protein interactions. Vaccinia-related kinase (VRK) proteins are Ser-Thr kinases that regulate several signal transduction pathways. In this work, we study the effect of VRK2 on MAPK signaling using breast cancer as a model. High levels of VRK2 inhibit EGF and ErbB2 activation of transcription by the serum response element (SRE). This effect is also detected in response to H-Ras(G12V) or B-Raf(V600E) oncogenes and is accompanied by a reduction in phosphorylated extracellular signal-regulated kinase (ERK) levels, p90RSK levels, and SRE-dependent transcription. Furthermore, VRK2 knockdown has the opposite effect, increasing the transcriptional response to stimulation with EGF and leading to increased levels of ERK phosphorylation. The molecular mechanism lies between MAPK/ERK kinase (MEK) and ERK, since MEK remains phosphorylated while ERK phosphorylation is blocked by VRK2A. This inhibition of the ERK signaling pathway is a consequence of a direct protein-protein interaction between VRK2A, MEK, and kinase suppressor of Ras 1 (KSR1). Identification of new correlations in human cancer can lead to a better understanding of the biology of individual tumors. ErbB2 and VRK2 protein levels were inversely correlated in 136 cases of human breast carcinoma. In ErbB2(+) tumors, there is a significant reduction in the VRK2 level, suggesting a role for VRK2A in ErbB2-MAPK signaling. Thus, VRK2 downregulation in carcinomas permits signal transmission through the MEK-ERK pathway without affecting AKT signaling, causing a signal imbalance among pathways that contributes to the phenotype of breast cancer.

Breast carcinomatous tumoral emboli can result from encircling lymphovasculogenesis rather than lymphovascular invasion.

The canonical view of the origin of tumor lymphovascular emboli is that they usually originate from lymphovascular invasion as part of a multistep metastatic process. Recent experimental evidence has suggested that metastasis can occur earlier than previously thought and we found evidence that tumor emboli formation can result from the short-circuiting step of encircling lymphovasculogenesis. Experimentally, we used a xenograft of human inflammatory breast cancer (MARY-X), a model that exhibited florid tumor emboli, to generate tumoral spheroids in vitro. In observational studies, we chose human breast carcinoma cases where there appeared to be a possible transition of in situ carcinoma to lymphovascular emboli without intervening stromal invasion. These cases were studied by morphometry as well as IHC with tumor proliferation (Ki-67) and adhesion (E-cadherin) markers, myoepithelial (p63), as well as endothelial (podoplanin [D2-40], CD31, VEGFR-3, Prox-1) markers. Unlabelled spheroids coinjected with either GFP or RFP-human myoepithelial cells or murine embryonal fibroblasts (MEFs) gave rise to tumors which exhibited GFP/RFP immunoreactivity within the cells lining the emboli-containing lymphovascular channels. In vitro studies demonstrated that the tumoral spheroids induced endothelial differentiation of cocultured myoepithelial cells and MEFs, measured by real time PCR and immunofluorescence. In humans, the in situ clusters exhibited similar proliferation, E-cadherin immunoreactivity and size as the tumor emboli (p =.5), suggesting the possibility that the latter originated from the former. The in situclusters exhibited a loss (50%-100%) of p63 myoepithelial immunoreactivity but not E-cadherin epithelial immunoreactivity. The tumor emboli were mainly present within lymphatic channels whose dual p63/CD31, p63/D2-40 and p63/VEGFR-3 and overall weak patterns of D2-40/CD31/VEGFR-3 immunoreactivities suggested that they represented immature and newly created vasculature derived from originally myoepithelial-lined ducts. Collectively both experimental as well as observational studies suggested the possibility that these breast cancer emboli resulted from encircling lymphovasculogenesis rather than conventional lymphovascular invasion.

Breast carcinomatous tumoral emboli can result from encircling lymphovasculogenesis rather than lymphovascular invasion.

The canonical view of the origin of tumor lymphovascular emboli is that they usually originate from lymphovascular invasion as part of a multistep metastatic process. Recent experimental evidence has suggested that metastasis can occur earlier than previously thought and we found evidence that tumor emboli formation can result from the short-circuiting step of encircling lymphovasculogenesis. Experimentally, we used a xenograft of human inflammatory breast cancer (MARY-X), a model that exhibited florid tumor emboli, to generate tumoral spheroids in vitro. In observational studies, we chose human breast carcinoma cases where there appeared to be a possible transition of in situ carcinoma to lymphovascular emboli without intervening stromal invasion. These cases were studied by morphometry as well as IHC with tumor proliferation (Ki-67) and adhesion (E-cadherin) markers, myoepithelial (p63), as well as endothelial (podoplanin [D2-40], CD31, VEGFR-3, Prox-1) markers. Unlabelled spheroids coinjected with either GFP or RFP-human myoepithelial cells or murine embryonal fibroblasts (MEFs) gave rise to tumors which exhibited GFP/RFP immunoreactivity within the cells lining the emboli-containing lymphovascular channels. In vitro studies demonstrated that the tumoral spheroids induced endothelial differentiation of cocultured myoepithelial cells and MEFs, measured by real time PCR and immunofluorescence. In humans, the in situ clusters exhibited similar proliferation, E-cadherin immunoreactivity and size as the tumor emboli (p =.5), suggesting the possibility that the latter originated from the former. The in situclusters exhibited a loss (50%-100%) of p63 myoepithelial immunoreactivity but not E-cadherin epithelial immunoreactivity. The tumor emboli were mainly present within lymphatic channels whose dual p63/CD31, p63/D2-40 and p63/VEGFR-3 and overall weak patterns of D2-40/CD31/VEGFR-3 immunoreactivities suggested that they represented immature and newly created vasculature derived from originally myoepithelial-lined ducts. Collectively both experimental as well as observational studies suggested the possibility that these breast cancer emboli resulted from encircling lymphovasculogenesis rather than conventional lymphovascular invasion.

Tumoral emboli and encircling lymphovasculogenesis

Indirect and circumstantial clinical evidence suggests that often occurs tumor progression. In stage breast cancer, for example, a significant percentage of patients exhibit circulating tumor cells (CTC's) and disseminated bone marrow tumor cells (DTC's) [[1, [2]. More direct experimental evidence for early metastasis has been observed studies of transgenic mice expressing a cancer-causing oncogene where the primary cancer on exists an in stage yet bone marrow metastases are discovered [3]. If indeed occurs early, then the rate-limiting steps which give rise to must also occur early. Some rate-limiting steps include both angiogenesis as well as lymphangiogenesis. However there have been alternate short-circuiting mechanisms proposed for these phenomenon including vasculogenic mimicry [4]. Another key rate-limiting step the metastatic process is tumoral embolic formation within lymphovascular channels. The canonical view of the origin of tumor lymphovascular emboli is that they directly originate from lymphovascular invasion. We found evidence, however that tumoral lymphovascular emboli can result from the short-circuiting step of encircling lymphovasculogenesis [5]. Experimentally, we used a xenograft of human inflammatory breast cancer (MARY-X), a model that exhibited florid tumor emboli, to generate tumoral spheroids vitro. In observational studies, we chose human breast carcinoma cases where there appeared to be a possible transition of situ carcinoma to lymphovascular emboli without intervening stromal invasion. These cases were studied by morphometry as well as IHC with tumor proliferation (Ki-67) and adhesion (E-cadherin) markers, myoepithelial (p63), as well as endothelial (podoplanin [D2-40], CD31, VEGFR-3, Prox-1) markers. Unlabelled spheroids coinjected with either GFP or RFP-human myoepithelial cells or murine embryonal fibroblasts (MEFs) gave rise to tumors which exhibited GFP/RFP immunoreactivity within the cells lining the emboli-containing lymphovascular channels. In vitro studies demonstrated that the tumoral spheroids induced endothelial differentiation of cocultured myoepithelial cells and MEFs, measured by real time PCR and immunofluorescence. In humans, the DCIS clusters exhibited similar proliferation, E-cadherin immunoreactivity and size as the tumor emboli (p =.5), suggesting the possibility that the latter originated from the former. The DCIS clusters exhibited a loss (50%-100%) of p63 myoepithelial immunoreactivity but not E-cadherin epithelial immunoreactivity. The tumor emboli were mainly present within lymphatic channels whose dual p63/CD31, p63/D2-40 and p63/VEGFR-3 and overall weak patterns of D2-40/CD31/VEGFR-3 immunoreactivities suggested that they represented immature and newly created vasculature derived from originally myoepithelial-lined ducts. Collectively both experimental as well as observational studies suggested the possibility that these breast cancer emboli resulted from encircling lymphovasculogenesis rather than conventional lymphovascular invasion. Encircling lymphovasculo-genesis as the mechanism of lymphovascular invasion represents a paradigm shift our thinking about this phenomenon. The lymphovascular embolus represents the phenomenon of the the where the arduous and complex task of constructing a ship within a bottle is replaced with the relatively effortless and simple job of building the bottle around the ship [5].

Aberrant Expression of X-Linked Genes RbAp46, Rsk4, and Cldn2 in Breast Cancer

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes.

Expression of somatostatin type 2A receptor correlates with estrogen receptor in human breast carcinoma.

Somatostatin type 2A receptor (sstr2A) has been shown to be directly involved in the transduction of antiproliferative effects and also to be the most predominant sstr subtype in human normal breast epithelium, as well as in human breast carcinoma. We investigated the immunoreactivity of sstr2A in 34 cases of human breast carcinoma and correlated these findings with the immunoreactivity of the estrogen receptor (ER), epidermal growth factor receptor (EGFR), transforming growth factor alpha (TGFalpha) and insulin like growth factor I (IGF-I). We detected sstr2A immunoreactivity in normal mammary tissue, and in 27 of 34 (79%) breast carcinomas. The sstr2A immunoreactivity was localized on the cellular membrane, however, weak cytoplasmic immunoreactivity was also observed. Sstr2A immunoreactivity was heterogenously distributed in the whole tumor section. There was a statistically significant correlation between sstr2A and ER immunoreactivity in the same tumor. No statistically significant correlation was found between sstr2A immunoreactivity and immunoreactivity for EGFR, TGFalpha and IGF-I or the patients' age.

Abnormal expression of MDM-2 in breast carcinomas.

MDM-2 is a cellular oncoprotein that binds to the p53 protein and abrogates its growth-suppressing function. At least seven MDM-2 mRNAs and five proteins (p90, p85, p76, p74, and p57) have been reported in tissue culture. MDM-2 gene amplification occurs in human sarcomas and high-grade gliomas. MDM-2 overexpression without gene amplification has been reported in leukemias and lymphomas. Here we report MDM-2 mRNA overexpression in 24 (73%) out of 33 cases of human breast carcinoma as compared with normal breast tissue. The MDM-2 overexpression was seen in the absence of MDM-2 gene amplification. MDM-2 protein expression was studied by western blot analysis in 21 of these cases of carcinoma. We found complete concordance between MDM-2 mRNA overexpression and MDM-2 protein levels. MDM-2 proteins were overexpressed in 15 of 21 breast carcinoma tissue samples but not in normal breast tissue controls. Ten of these fifteen cases overexpressed MDM-2 p57 protein, two cases overexpressed both p57 and p90, and three cases overexpressed only p90. MDM-2 overexpression was confirmed by immunohistochemistry. p53 overexpression was also studied by immunohistochemistry, 69% of breast carcinomas that overexpressed the MDM-2 mRNA had detectable nuclear p53 protein. These findings demonstrate that MDM-2 oncoprotein expression is altered in primary human breast carcinomas at both mRNA and protein levels. In addition, our results suggest that MDM-2 p57 protein represents the main MDM-2 protein altered in breast carcinomas.

Expression of mRNA for gelatinase A and TIMP-2 in cell cultures and tissue samples derived from breast and lung carcinomas

The aim of this study was to determine how closely expression of MMP-2 and TIMP-2 is associated with poor patient prognosis in breast and lung cancer. mRNA levels of MMP-2 and TIMP-2 were examined on dot blots in 34 cases of human breast carcinoma and in 13 cases of lung carcinoma. In breast carcinomas we found that high MMP-2 and/or low TIMP-2 expression did not consistently correspond to a poor clinical situation, while high MMP-2/TIMP-2 ratios were also found in subjects whose clinical status was relatively favorable. Statistical analysis did not show any significant association between MMP-2/TIMP-2 and the tumor markers examined. Regarding lung carcinomas, we did not find a correlation between the ratio and poor prognosis, whereas in lung carcinoma cells in vitro we found an enhancement in MMP-2 production. A possible explanation for these results is that in vivo, as opposed to in vitro, it appears that the stromal cells of the tumor and not the tumor cell itself, produce MMP-2.

Distribution of thrombospondin and integrin alpha V in DCIS, invasive ductal and lobular human breast carcinomas. Analysis by electron microscopy.

The ultrastructural distribution of thrombospondin (TSP) and its cell surface receptor, integrin alpha V, was studied in two cases of human breast carcinoma: one of ductal carcinoma in situ (DCIS) with an invasive component, and one of invasive lobular carcinoma. In DCIS, moderate immunolabelling for TSP and integrin alpha V was observed in the rough endoplasmic reticulum and at the plasma membrane of intraductal carcinoma cells. TSP was also associated with extracellular matrix collagen fibrils surrounding in situ carcinoma cells. In the invasive part of this ductal carcinoma, most of the malignant cells were negative for TSP, while integrin alpha V was moderately expressed in these cells. In sharp contrast, typical strands of invasive lobular carcinoma cells in "Indian file" showed moderate TSP immunostaining in the rough endoplasmic reticulum and strong immunoreactivity for TSP at the plasma membrane and in the extracellular matrix. Moderate to strong immunoreactivity for integrin alpha V was also observed in invasive lobular carcinoma cells. Because of the role of TSP during cancer cell invasion, the different expression patterns of TSP in invasive ductal versus lobular carcinoma may well reflect biological differences between these two types of breast carcinoma and could account for the peculiar diffuse invasive behaviour of breast lobular carcinoma cells.

A new immunohistochemical antibody for the assessment of estrogen receptor status on routine formalin-fixed tissue samples

We describe a method for the immunocytochemical assessment of estrogen receptor (ER) status on routinely processed, formalinfixed tissue using a recently developed commercially available monoclonal antibody (Dako 1D5) with a microwave antigen retrieval technique. A series of 90 cases of human breast carcinoma was analyzed and the staining was assessed using a semiquantitative microscopic scoring method and by assessment of the percentage of nuclei showing positive staining. The results were compared with assessment using another commercially available antibody (Abbott H222) and with clinical response to tamoxifen therapy. Direct comparison of the paired sets of H scores and the percentage of positively stained nuclei using the two different techniques showed a significant correlation. By assigning an arbitrary cut-off for positivity of H score = 50, assessment of ER status using DAKO 1D5 antibody was found to correlate with response to tamoxifen therapy with a sensitivity of 90% and a specificity of 51%, similar to values in previous studies using other methods. We conclude that assessment of ER status using this new antibody and technique gives accurate results on routinely processed, formalin-fixed tissue and may be used as an alternative to other methods.

Expression of epidermal growth factor receptor (proto-oncogene c-erbB-1) and estrogen receptor in human breast carcinoma. An immunocytochemical study of 70 cases.

In vitro studies have shown that growth factors may mediate the growth stimulatory effect of estrogen in hormone-dependent human breast carcinomas while the constitutive expression of same growth factors might by-pass the need for estrogenic stimulus in hormone-independent neoplasms. We have performed immunocytochemical analysis of the expression of epidermal growth factor receptor (EGF-R or proto-oncogen c-erbB-1) and estrogen receptor (ER) in 70 cases of human breast carcinoma. We found an inverse relationship between the expression of EGF-R and ER (Kendall's tau b = -0.1997, P < 0.03), which prompts us to conclude that ER(-) breast carcinomas may grow in a hormone-independent manner through the over-expression of the proto-oncogene c-erbB-1, which is the receptor for epidermal (EGF) and alpha transforming (TGFalfa) growth factors.

61 Chromosome banding analysis of 26 cases of human breast carcinoma: JM Yang, WS Dalton, A Leibovitz, FH Thompson, JM Trent. University of Arizona, Cancer Center, Tucson, AZ 85724

61 Chromosome banding analysis of 26 cases of human breast carcinoma: JM Yang, WS Dalton, A Leibovitz, FH Thompson, JM Trent. University of Arizona, Cancer Center, Tucson, AZ 85724

Low temperature backscattered electron imaging in the study of human tissues.

A scanning electron microscopic technique for the examination of bulk, fresh, hydrated human tissue is described. Samples of fresh tissue are frozen in liquid nitrogen against a mirror-finished copper block and planed in a cryoultramicrotome before transfer to a low temperature scanning electron microscope. After sublimation of water from the specimen surface, the tissue is examined in secondary electron and backscattered electron modes. Adjacent pieces of tissue, and those retrieved after backscattered electron observation, can be readily prepared for and examined by light and by conventional transmission electron microscopy. The method has been tested with multiple blocks taken from 6 cases of human breast carcinoma. In the backscattered electron mode, the infiltrating columns of neoplastic cells can be distinguished from mammary adipose and fibrous tissue. Within a carcinoma, the collagenous stroma, carcinoma cells, and perivascular and perineural infiltrates can be identified. These features have been contrasted with those obtained by light microscopy, by low temperature scanning, and by transmission electron microscopy. This use of backscattered electron imaging for the investigation of unfixed hydrated tissue offers the possibility that the technique could be of considerable value in the microscopy of very small samples in which, because of a need for subsequent biochemical, histochemical, and immunologic investigation, fixation and dehydration are to be avoided.

A histochemical and ultrastructural study of human breast carcinomas with a view to their classification by cell of origin

We have studied 450 cases of human breast carcinoma at the histochemical and ultrastructural level. The breast biopsies were obtained from Canada, Mexico and Dominican Republic. This study was undertaken to evaluate the prognostic significance of classification of breast tumors, proposed in 1971 by Murad, based on their cell of origin. Under this classification, ATPase positive tumors represent those of myoepithelial origin, while the remainder are designated ductal or ductular based upon defined ultrastructural characteristics. We observed a distribution of tumor types essentially similar to that reported by Murad, and no significant differences were observed between the contributing centers. No correlation was observed between this classification and the histological classification described by Stewart. We found no evidence that ATPase positive tumors were myoepithelial in origin. They showed histochemical similarities to ductal and ductular tumors, and had features indicating their secretory nature. Estrogen receptor levels were measured in 20 cases, and did not reveal any correlation with tumor type. An interesting feature requiring further investigation was the high incidence of endocrine-like secretory granules in all histological types of tumors studied.