Alterations of Smad signaling in human breast carcinoma are associated with poor outcome: a tissue microarray study.

Based largely on studies of cell lines in vitro and of transgenic mouse models, disruptions of transforming growth factor (TGF) beta signaling are thought to contribute to the development and progression of human breast cancer. However, whether and how TGF-beta signaling becomes disrupted during human breast cancer development in vivo remains largely unknown. To address this question, we have compared the patterns of expression and activation of the postreceptor components of the TGF-beta signaling pathway, the so-called Smads, in human breast cancer cell lines with those in breast carcinoma specimens. None of the breast carcinoma cell lines were growth arrested by TGF-beta in vitro. Each of the tumor cell lines expressed normal levels of Smad2 and -3. Moreover, TGF-beta treatment induced phosphorylation of Smad2 (Smad2P) in each of these lines, except those that lacked TGF-beta type II receptors. Moreover, only one of the cell lines failed to express Smad4. Among 456 cases of human breast carcinoma assembled in tissue microarrays, the majority (92%) expressed Smad2, Smad2P, as well as Smad4, indicating their ability to proliferate within a microenvironment that contains bioactive TGF-beta. Thirty cases (6.6%) failed to express Smad2P, suggesting the loss of TGF-beta receptor signaling. Nine cases (2%) failed to express Smad4, and 3 of these also failed to express Smad2P. Thus, the phenotypes of breast tumors in vivo paralleled that of human breast cancer cell lines in terms of Smad2P and Smad4 expression. Loss of Smad signaling was not associated with any particular histological subtype, histological or nuclear grade, estrogen- or progesterone receptor expression, or HER2/neu expression. Loss of Smad4 was inversely correlated with the presence of axillary lymph node metastases. Most importantly, among patients with stage II breast cancer, lack of Smad2P expression in the tumor was strongly associated with shorter overall survival. Finally, analysis of a small cohort of hereditary breast cancers failed to reveal any association between BRCA1 or BRCA2 genotype and alterations in Smad signaling.

[1]  T. Stukel,et al.  Immunohistochemical staining for transforming growth factor beta 1 associates with disease progression in human breast cancer. , 1992, Cancer Research.

[2]  A. Geiser,et al.  Targeting expression of a transforming growth factor beta 1 transgene to the pregnant mammary gland inhibits alveolar development and lactation. , 1993, The EMBO journal.

[3]  M. Reiss,et al.  Transforming growth factor beta type I receptor kinase mutant associated with metastatic breast cancer. , 1998, Cancer research.

[4]  P. Dijke,et al.  DPC4 (SMAD4) mediates transforming growth factor-β1 (TGF-β1) induced growth inhibition and transcriptional response in breast tumour cells , 1997, Oncogene.

[5]  Irene L Andrulis,et al.  MADR2 Maps to 18q21 and Encodes a TGFβ–Regulated MAD–Related Protein That Is Functionally Mutated in Colorectal Carcinoma , 1996, Cell.

[6]  M. Barcellos-Hoff,et al.  Transforming growth factor-β and breast cancer: Mammary gland development , 2000, Breast Cancer Research.

[7]  L. Wakefield,et al.  Transforming growth factor-β1 is a new form of tumor suppressor with true haploid insufficiency , 1998, Nature Medicine.

[8]  E. Kaplan,et al.  Nonparametric Estimation from Incomplete Observations , 1958 .

[9]  P J Catalano,et al.  Molecular predictors of survival after adjuvant chemotherapy for colon cancer. , 2001, The New England journal of medicine.

[10]  Kathleen R. Cho,et al.  A transforming growth factor beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability. , 1995, Cancer research.

[11]  Z. Hall.  Cancer , 1906, The Hospital.

[12]  E. L. Springer,et al.  Two syngeneic cell lines from human breast tissue: the aneuploid mammary epithelial (Hs578T) and the diploid myoepithelial (Hs578Bst) cell lines. , 1977, Journal of the National Cancer Institute.

[13]  R. Strange,et al.  Apoptotic cell death and tissue remodelling during mouse mammary gland involution. , 1992, Development.

[14]  C. Zuiani,et al.  Human mammary gland and breast carcinoma contain immunoreactive inhibin/activin subunits: evidence for a secretion into cystic fluid. , 1999, European journal of endocrinology.

[15]  M. Reiss,et al.  Transforming growth factor-β in breast cancer: A working hypothesis , 1997, Breast Cancer Research and Treatment.

[16]  B. Hogan,et al.  Inhibition of mammary duct development but not alveolar outgrowth during pregnancy in transgenic mice expressing active TGF-beta 1. , 1993, Genes & development.

[17]  J. Kononen,et al.  Tissue microarrays for high-throughput molecular profiling of tumor specimens , 1998, Nature Medicine.

[18]  L. Wakefield,et al.  Transgenic Mice Overexpressing a Dominant-negative Mutant Type II Transforming Growth Factor β Receptor Show Enhanced Tumorigenesis in the Mammary Gland and Lung in Response to the Carcinogen 7,12-Dimethylbenz-[a]-anthracene , 1997 .

[19]  T. Chen,et al.  In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. , 1977, Experimental cell research.

[20]  R. Hesketh,et al.  Inhibiting mutations in the transforming growth factor beta type 2 receptor in recurrent human breast cancer. , 2001, Cancer research.

[21]  M. Radu,et al.  Establishment and characterization of a cell line of human breast carcinoma origin. , 1979, European journal of cancer.

[22]  A. Greenberg,et al.  Immunocytochemical localization of secreted transforming growth factor-beta 1 to the advancing edges of primary tumors and to lymph node metastases of human mammary carcinoma. , 1993, The American journal of pathology.

[23]  J. Massagué.  TGF-beta signal transduction. , 1998, Annual review of biochemistry.

[24]  Simpson,et al.  Loss of expression of transforming growth factor beta type II receptor correlates with high tumour grade in human breast in‐situ and invasive carcinomas , 2000, Histopathology.

[25]  M. Sporn,et al.  Type beta transforming growth factor: a bifunctional regulator of cellular growth. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[26]  B. Weissman,et al.  BALB and kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines , 1983, Cell.

[27]  M. Reiss,et al.  Smad protein expression and activation in transforming growth factor-beta refractory human squamous cell carcinoma cells. , 2001, Oncology research.

[28]  W. Dupont,et al.  Transforming Growth Factor-β and Breast Cancer Risk in Women With Mammary Epithelial Hyperplasia. , 1999, Journal of the National Cancer Institute.

[29]  S. Kern,et al.  Dpc4 transcriptional activation and dysfunction in cancer cells. , 1998, Cancer research.

[30]  J. Keski‐Oja,et al.  Latency, activation, and binding proteins of TGF‐β , 2001, Microscopy research and technique.

[31]  J. Willson,et al.  Expression of transforming growth factor β type II receptor leads to reduced malignancy in human breast cancer MCF-7 cells , 1994 .

[32]  A. Roberts,et al.  Characterization of Functional Domains within Smad4/DPC4* , 1997, The Journal of Biological Chemistry.

[33]  A. Okamoto,et al.  Overexpression of human cyclin D1 reduces the transforming growth factor beta (TGF-beta) type II receptor and growth inhibition by TGF-beta 1 in an immortalized human esophageal epithelial cell line. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[34]  H. Hollema,et al.  Structural alterations of transforming growth factor‐β receptor genes in human cervical carcinoma , 1999, International journal of cancer.

[35]  J. Massagué,et al.  Partnership between DPC4 and SMAD proteins in TGF-β signalling pathways , 1996, Nature.

[36]  R. Coffey,et al.  Mammary tumor suppression by transforming growth factor beta 1 transgene expression. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  R. Coombes,et al.  Inhibitory effects of activin on the growth and morpholgenesis of primary and transformed mammary epithelial cells. , 1996, Cancer research.

[38]  R. Hruban,et al.  Loss of expression of Dpc4 in pancreatic intraepithelial neoplasia: evidence that DPC4 inactivation occurs late in neoplastic progression. , 2000, Cancer research.

[39]  R. Walker,et al.  Transforming growth factor beta 1 in ductal carcinoma in situ and invasive carcinomas of the breast. , 1992, European journal of cancer.

[40]  Kathleen R. Cho,et al.  DPC4 gene in various tumor types. , 1996, Cancer research.

[41]  B. Groner,et al.  Transforming Growth Factor-β Inhibits Lactogenic Hormone Induction of β-Casein Expression in HC11 Mouse Mammary Epithelial Cells , 1990 .

[42]  Jiri Bartek,et al.  Cyclin D1 protein expression and function in human breast cancer , 1994, International journal of cancer.

[43]  S. M. Svoboda-Newman,et al.  Comparison of Methods for Extracting DNA from Formalin‐Fixed Paraffin Sections for Nonisotopic PCR , 1996, Diagnostic molecular pathology : the American journal of surgical pathology, part B.

[44]  R. Laucirica,et al.  Transforming Growth Factor‐ β Type II Receptor Is Infrequently Expressed in Human Breast Cancer , 1996 .

[45]  J. Willson,et al.  Expression of transforming growth factor‐β receptor type II and tumorigenicity in human breast adenocarcinoma MCF‐7 cells , 1998 .

[46]  A. Long,et al.  A human cell line from a pleural effusion derived from a breast carcinoma. , 1973, Journal of the National Cancer Institute.

[47]  K. Kinzler,et al.  Microsatellite instability and mutations of the transforming growth factor beta type II receptor gene in colorectal cancer. , 1995, Cancer research.

[48]  D. Medina,et al.  Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[49]  T. Soussi,et al.  Epithelial HBL-100 cell line derived from milk of an apparently healthy woman harbours SV40 genetic information. , 1985, Experimental cell research.

[50]  M. Reiss,et al.  Transforming growth factor beta type I receptor kinase mutant associated with metastatic breast cancer. , 1998, Cancer research.

[51]  A. Sparks,et al.  Immunohistochemical labeling for Dpc4 mirrors genetic status in pancreatic adenocarcinomas: A new marker of DPC4 inactivation , 2000 .

[52]  K. Kinzler,et al.  Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. , 1995, Science.

[53]  E Y LASFARGUES,et al.  Cultivation of human breast carcinomas. , 1958, Journal of the National Cancer Institute.

[54]  F. Pouliot,et al.  Expression profile of agonistic Smads in human breast cancer cells: Absence of regulation by estrogens , 1999, International journal of cancer.

[55]  E. Gaffney.  A cell line (HBL-100) established from human breast milk , 2004, Cell and Tissue Research.

[56]  J. Massagué,et al.  TGF- SIGNAL TRANSDUCTION , 1998 .

[57]  J. Bartek,et al.  Amplification and overexpression of cyclin D1 in breast cancer detected by immunohistochemical staining. , 1994, Cancer research.

[58]  A. Green,et al.  Expression and secretion of TGF-beta isoforms and expression of TGF-beta-receptors I, II and III in normal and neoplastic human breast. , 1999, International journal of oncology.

[59]  K. Kinzler,et al.  Mutational inactivation of transforming growth factor beta receptor type II in microsatellite stable colon cancers. , 1999, Cancer research.

[60]  J. Massagué,et al.  Defective repression of c-myc in breast cancer cells: A loss at the core of the transforming growth factor beta growth arrest program. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[61]  S. Kern,et al.  Transforming growth factor‐β responsiveness in DPC4/SMAD4‐null cancer cells , 1999 .

[62]  Adriana,et al.  Resistance to Transforming Growth Factor 1 and Activin Due to Reduced Receptor Expression in Human Breast Tumor Cell Lines ’ , 2005 .

[63]  S. O’Brien,et al.  Establishment and characterization of three new continuous cell lines derived from human breast carcinomas. , 1978, Cancer research.

[64]  P. V. van Diest,et al.  Expression of growth factors, growth inhibiting factors, and their receptors in invasive breast cancer. I: An inventory in search of autocrine and paracrine loops , 1998, The Journal of pathology.

[65]  M. Sporn,et al.  Localization of transforming growth factor-beta isotypes in lesions of the human breast. , 1992, Human pathology.

[66]  D. Rimm,et al.  Validation of Tissue Microarray Technology in Breast Carcinoma , 2000, Laboratory Investigation.