A survey of health care professionals and oncology patients at the McGill University Health Centre reveals enthusiasm for establishing a postmortem rapid tissue donation program.

Background: In the early developmental phase of a postmortem rapid tissue donation (rtd) program for patients with metastatic cancer, we surveyed health care professionals (hcps) and oncology patients at the McGill University Health Centre (muhc) to assess their knowledge and attitudes pertaining to rtd from metastatic cancer patients for research purposes. Methods: A 23-item survey was developed and distributed to hcps at tumour board meetings, and a related 26-item survey was developed and distributed to oncology patients at the muhc Cedars Cancer Centre. Results: The survey attracted participation from 73 hcps, including 37 attending physicians, and 102 oncology patients. Despite the fact that 88% of hcps rated their knowledge of rtd as none or limited, 42% indicated that they would feel comfortable discussing rtd with their cancer patients. Of the responding hcps, 67% indicated that their current knowledge of rtd would affect their decision to discuss such a program with patients, which implies the importance of education for hcps to facilitate enrolment of patients into a rtd program. Of responding patients, 78% indicated that they would not be uncomfortable if their doctor discussed rtd with them, and 61% indicated that they would like it if their doctor were to discuss rtd with them. The hcps and patients felt that the best time for patients to be approached about consenting to a rtd program would be at the transition to palliative care when no treatment options remain. Conclusions: At the muhc, hcps and patients are generally enthusiastic about adopting a rtd program for patients with metastatic cancer. Education of hcps and patients will be an important determinant of the program's success.

LKB1 deficiency in T cells promotes the development of gastrointestinal polyposis.

Germline mutations in STK11, which encodes the tumor suppressor liver kinase B1 (LKB1), promote Peutz-Jeghers syndrome (PJS), a cancer predisposition syndrome characterized by the development of gastrointestinal (GI) polyps. Here, we report that heterozygous deletion of Stk11 in T cells (LThet mice) is sufficient to promote GI polyposis. Polyps from LThet mice, Stk11+/- mice, and human PJS patients display hallmarks of chronic inflammation, marked by inflammatory immune-cell infiltration, signal transducer and activator of transcription 3 (STAT3) activation, and increased expression of inflammatory factors associated with cancer progression [interleukin 6 (IL-6), IL-11, and CXCL2]. Targeting either T cells, IL-6, or STAT3 signaling reduced polyp growth in Stk11+/- animals. Our results identify LKB1-mediated inflammation as a tissue-extrinsic regulator of intestinal polyposis in PJS, suggesting possible therapeutic approaches by targeting deregulated inflammation in this disease.

The Cdc42/Rac1 regulator CdGAP is a novel E-cadherin transcriptional co-repressor with Zeb2 in breast cancer.

The loss of E-cadherin causes dysfunction of the cell-cell junction machinery, which is an initial step in epithelial-to-mesenchymal transition (EMT), facilitating cancer cell invasion and the formation of metastases. A set of transcriptional repressors of E-cadherin (CDH1) gene expression, including Snail1, Snail2 and Zeb2 mediate E-cadherin downregulation in breast cancer. However, the molecular mechanisms underlying the control of E-cadherin expression in breast cancer progression remain largely unknown. Here, by using global gene expression approaches, we uncover a novel function for Cdc42 GTPase-activating protein (CdGAP) in the regulation of expression of genes involved in EMT. We found that CdGAP used its proline-rich domain to form a functional complex with Zeb2 to mediate the repression of E-cadherin expression in ErbB2-transformed breast cancer cells. Conversely, knockdown of CdGAP expression led to a decrease of the transcriptional repressors Snail1 and Zeb2, and this correlated with an increase in E-cadherin levels, restoration of cell-cell junctions, and epithelial-like morphological changes. In vivo, loss of CdGAP in ErbB2-transformed breast cancer cells impaired tumor growth and suppressed metastasis to lungs. Finally, CdGAP was highly expressed in basal-type breast cancer cells, and its strong expression correlated with poor prognosis in breast cancer patients. Together, these data support a previously unknown nuclear function for CdGAP where it cooperates in a GAP-independent manner with transcriptional repressors to function as a critical modulator of breast cancer through repression of E-cadherin transcription. Targeting Zeb2-CdGAP interactions may represent novel therapeutic opportunities for breast cancer treatment.

The role of claudins in cancer metastasis.

TJs are large intercellular adhesion complexes that maintain cell polarity in normal epithelia and endothelia. During the metastatic process, TJs must be 'loosened' or dismantled in cancer cells to enable migration and dissemination. Diminished TJ integrity must also occur within endothelial cells to allow intravasation and extravasation of cancer cells across endothelial barriers. Claudins are critical components of TJs, forming homo- and heteromeric interactions between the adjacent cells, which have been implicated as key modulators of carcinogenesis and metastasis. Numerous epithelial-derived cancers display altered claudin expression patterns and certain claudins can now be used as biomarkers to predict patient prognosis. Moreover, claudins have been functionally implicated in numerous steps of the metastatic cascade. The distinct roles played by claudins during the cancer progression to metastatic disease are just starting to be elucidated. A more complete understanding of the mechanisms through which claudins augment cancer metastasis is required to develop new therapeutic agents against this family of proteins. In this review, we will summarize the relationship between the claudin expression and clinical outcomes in diverse cancers, discuss tumor intrinisic roles through which claudins regulate metastasis and explore claudin-mediated functions within stromal cells that influence the metastatic process. Finally, we will consider possible strategies for targeting claudins that have the potential to improve the management of metastatic cancer.

GPNMB cooperates with neuropilin-1 to promote mammary tumor growth and engages integrin α5ß1 for efficient breast cancer metastasis.

Glycoprotein nmb (GPNMB) promotes breast tumor growth and metastasis and its expression in tumor epithelium correlates with poor prognosis in breast cancer patients. Despite its biological and clinical significance, little is known regarding the molecular mechanisms engaged by GPNMB. Herein, we show that GPNMB engages distinct functional domains and mechanisms to promote primary tumor growth and metastasis. We demonstrate that neuropilin-1 (NRP-1) expression is increased in breast cancer cells that overexpress GPNMB. Interestingly, the GPNMB-driven increase in NRP-1 expression potentiated vascular endothelial growth factor signaling in breast cancer cells and was required for the growth, but not metastasis, of these cells in vivo. Interrogation of RNAseq data sets revealed a positive correlation between GPNMB and NRP-1 levels in human breast tumors. Furthermore, we ascribe pro-growth and pro-metastatic functions of GPNMB to its ability to bind α5ß1 integrin and increase downstream signaling in breast cancer cells. We show that GPNMB enhances breast cancer cell adhesion to fibronectin, increases α5ß1 expression and associates with this receptor through its RGD motif. GPNMB recruitment into integrin complexes activates Src and Fak signaling pathways in an RGD-dependent manner. Importantly, both the RGD motif and cytoplasmic tail of GPNMB are required to promote primary mammary tumor growth; however, only mutation of the RGD motif impaired the formation of lung metastases. Together, these findings identify novel and distinct molecular mediators of GPNMB-induced breast cancer growth and metastasis.

Claudin-2 is selectively enriched in and promotes the formation of breast cancer liver metastases through engagement of integrin complexes.

The liver represents the third most frequent site of metastasis in patients with breast cancer. We performed in vivo selection using 4T1 breast cancer cells to identify genes associated with the liver metastatic phenotype. Coincident with the loss of numerous tight-junctional proteins, we observe claudin-2 overexpression, specifically in liver-aggressive breast cancer cells. We further demonstrate that claudin-2 is both necessary and sufficient for the ability of 4T1 breast cancer cells to colonize and grow in the liver. The liver-aggressive breast cancer cells display a claudin-2-mediated increase in their ability to adhere to extracellular matrix (ECM) components, such as fibronectin and type IV collagen. Claudin-2 facilitates these cell/matrix interactions by increasing the cell surface expression of α(2)ß(1)- and α(5)ß(1)-integrin complexes in breast cancer cells. Indeed, claudin-2-mediated adhesion to fibronectin and type IV collagen can be blocked with neutralizing antibodies that target α(5)ß(1) and α(2)ß(1) complexes, respectively. Immunohistochemical analyses reveal that claudin-2, although weakly expressed in primary human breast cancers, is readily detected in all liver metastasis samples examined to date. Together, these results uncover novel roles for claudin-2 in promoting breast cancer adhesion to the ECM and define its importance during breast cancer metastasis to the liver.

CdGAP is required for transforming growth factor ß- and Neu/ErbB-2-induced breast cancer cell motility and invasion.

RhoA, Rac1 and Cdc42, the best-characterized members of the Rho family of small GTPases, are critical regulators of many cellular activities. Cdc42 GTPase-activating protein (CdGAP) is a serine- and proline-rich RhoGAP protein showing GAP activity against both Cdc42 and Rac1 but not RhoA. CdGAP is phosphorylated downstream of the MEK-ERK (extracellular signal-regulated kinase) pathway in response to serum and is required for normal cell spreading and polarized lamellipodia formation. In this study, we found that CdGAP protein and mRNA levels are highly increased in mammary tumor explants expressing an activated Neu/ErbB-2 (Neu-NT) receptor. In response to transforming growth factor-ß (TGFß) stimulation, Neu-NT-expressing mammary tumor explants demonstrate a clear induction in cell motility and invasion. We show that downregulation of CdGAP expression by small interfering RNA abrogates the ability of TGFß to induce cell motility and invasion of Neu-NT-expressing mammary tumor explants. However, it has no effect on TGFß-mediated cell adhesion on type 1 collagen and fibronectin. Interestingly, protein expression of E-Cadherin is highly increased in Neu-NT-expressing mammary tumor explants depleted of CdGAP. In addition, complete loss of E-Cadherin expression is not observed in CdGAP-depleted cells during TGFß-mediated epithelial to mesenchymal transition. Downregulation of the CdGAP expression also decreases cell proliferation of Neu-NT-expressing mammary tumor explants independently of TGFß. Rescue analysis using re-expression of various CdGAP deletion-mutant proteins revealed that the proline-rich domain (PRD) but not the GAP domain of CdGAP is essential to mediate TGFß-induced cell motility and invasion. Finally, we found that TGFß induces the expression and phosphorylation of CdGAP in mammary epithelial NMuMG cells. Taken together, these studies identify CdGAP as a novel molecular target in TGFß signaling and implicate CdGAP as an essential component in the synergistic interaction between TGFß and Neu/ErbB-2 signaling pathways in breast cancer cells.

Transcription factor regulatory networks in mammary epithelial development and tumorigenesis.

The mouse mammary gland is composed of three epithelial cell types, which include ductal, alveolar and myoepithelial cells. A hierarchy in which the mammary stem cell compartment gives rise to progressively restricted progenitors that ultimately form the luminal (ductal and alveolar) and myoepithelial lineages is now emerging. Although very little is known about the mechanisms controlling the differentiation of the myoepithelial cell lineage, a growing body of work reveals that the luminal cell fate is specified by a network of transcription factors. The precise roles of specific transcription factors in promoting differentiation of luminal progenitors into ductal or alveolar cells are now being elucidated. This review will discuss the importance of these recent observations and place them within the context of other transcription factor networks involved in mammary gland development and tumorigenesis.

The Ste20-like kinase SLK is required for ErbB2-driven breast cancer cell motility.

The Ste20-like kinase, SLK, is involved in the control of cell motility through its effects on actin reorganization and focal adhesion turnover. Here we investigated the role of SLK in chemotaxis downstream of the tyrosine kinase receptor, HER2/ErbB2/Neu, which is frequently overexpressed in human breast cancers. Our results show that SLK is required for the efficient cell migration of human and mouse mammary epithelial cell lines in the presence of the Neu activator, heregulin, as a chemoattractant. SLK activity is stimulated by heregulin treatment or by overexpression of activated Neu. Phosphorylation of tyrosine 1201 or tyrosines 1226/7 on Neu is a key event for SLK activation and cell migration, and cancer cell invasion mediated by these tyrosines is inhibited by kinase-inactive SLK. Signaling pathway inhibitors show that Neu-mediated SLK activation is dependent on MEK, PI3K, PLCgamma and Shc signaling. Furthermore, heregulin-stimulated SLK activity requires signals from the focal adhesion proteins, FAK and src. Finally, phospho-FAK analysis shows that SLK is required for Neu-dependent focal adhesion turnover. Together, these studies define an interaction between Neu and SLK signaling in the regulation of cancer cell motility.

GATA3 inhibits breast cancer growth and pulmonary breast cancer metastasis.

The loss of expression of the transcription factor GATA3 in breast tumors has been linked to aggressive tumor development and poor patient survival. In the present work, we address potential roles for GATA3 in breast tumor lung metastasis and progression. Using an aggressive breast cancer cell line, which metastasizes specifically to the lung, we show that GATA3 expression results in reduced tumor outgrowth in the mammary fat pad and lower lung metastatic burden in nude mice. Specifically, GATA3 expression inhibits breast cancer cell expansion inside the lung parenchyma. This phenotype correlates with the ability of GATA3 to negatively regulate the expression of several genes that promote breast cancer lung metastasis (ID1/-3, KRTHB1, LY6E and RARRES3). Conversely, the expression of genes encoding known inhibitors of lung metastasis (DLC1 (deleted in liver cancer 1) and PAEP (progestagen-associated endometrial protein)) is upregulated by GATA3. These data correlate with microarray data from human breast cancer patients, showing a strong correlation between high GATA3 expression and absence of metastases specifically to the lungs. We conclude that GATA3 inhibits primary breast tumor outgrowth and reduces lung metastatic burden by regulating key genes involved in metastatic breast tumor progression.

Transforming growth factor-beta1 is the predominant isoform required for breast cancer cell outgrowth in bone.

Transforming growth factor (TGF)-beta signaling is a potent modulator of the invasive and metastatic behavior of breast cancer cells. Indeed, breast tumor responsiveness to TGF-beta is important for the development of osteolytic bone metastases. However, the specific TGF-beta isoforms that promote breast cancer outgrowth in bone is unknown. We demonstrate that expression of a TGF-beta ligand trap, which neutralizes TGF-beta1 and TGF-beta3, in MDA-MB-231 breast cancer cells diminished their outgrowth in bone and reduced the severity of osteolytic lesion formation when compared with controls. We further show that a reduction or loss of TGF-beta1 expression within the bone microenvironment of TGF-beta1+/- and TGF-beta1-/- mice significantly reduced the incidence of breast tumor outgrowth compared with wild-type animals. Interestingly, those tumors capable of growing within the tibiae of TGF-beta1-deficient mice had upregulated expression of all three TGF-beta isoforms. Finally, breast cancer cells expressing the TGF-beta ligand trap showed a pronounced reduction in their ability to form osteolytic lesions when injected into the tibiae of TGF-beta1+/- mice. Thus, our studies show that both host- and tumor-derived TGF-beta expression plays a critical role during the establishment and outgrowth of breast cancer cells in bone.

Identifying site-specific metastasis genes and functions.

Metastasis is a multistep and multifunctional biological cascade that is the final and most life-threatening stage of cancer progression. Understanding the biological underpinnings of this complex process is of extreme clinical relevance and requires unbiased and comprehensive biological scrutiny. In recent years, we have utilized a xenograft model of breast cancer metastasis to discover genes that mediate organ-specific patterns of metastatic colonization. Examination of transcriptomic data from cohorts of primary breast cancers revealed a subset of site-specific metastasis genes that are selected for early in tumor progression. High expression of these genes predicts the propensity for lung metastasis independently of several classic markers of poor prognosis. These genes fulfill dual functions-enhanced primary tumorigenicity and augmented organ-specific metastatic activity. Other metastasis genes fulfill functions specialized for the microenvironment of the metastatic site and are consequently not selected for in primary tumors. These findings improve our understanding of metastatic progression, facilitate the interpretation of primary tumor gene expression data, and open several important possibilities for future clinical application.

Mammary gland neoplasia: insights from transgenic mouse models.

Current theories of breast cancer progression have been greatly influenced by the development and refinement of mouse transgenic and gene targeting technologies. Early transgenic mouse models confirmed the involvement of oncogenes, previously implicated in human breast cancer, by establishing a causal relationship between overexpression or activation of these genes and mammary tumorigenesis. More recently, the importance of genes located at sites of loss of heterozygosity in human breast cancer have been examined in mice by their targeted disruption via homologous recombination. The union of these two approaches allows the generation of complex animal models that more accurately reflect the multistep nature of human breast cancer. This review will examine how the study of transgenic mice has increased our understanding of the molecular events responsible for oncogenic transformation of the mammary gland. BioEssays 22:554-563, 2000.

Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis.

The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway.

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.

Accelerated mammary tumor development in mutant polyomavirus middle T transgenic mice expressing elevated levels of either the Shc or Grb2 adapter protein.

The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling.

Elevated expression of activated forms of Neu/ErbB-2 and ErbB-3 are involved in the induction of mammary tumors in transgenic mice: implications for human breast cancer.

To assess the importance of Neu activation during mammary tumorigenesis, altered receptors harboring in-frame deletions within the extracellular domain were expressed in transgenic mice. Females from several independent lines develop multiple mammary tumors that frequently metastasize to the lung. Tumor progression in these strains was associated with elevated levels of tyrosine-phosphorylated Neu and ErbB-3. Consistent with these observations, a survey of primary human breast tumors revealed frequent co-expression of both erbB-2 and erbB-3 transcripts. The ability of altered Neu receptors to induce mammary tumorigenesis in transgenic mice prompted us to examine whether similar mutations occurred in ErbB-2 during human breast cancer progression. Interestingly, an alternatively spliced form of erbB-2, closely resembling spontaneous activated forms of neu, was detected in human breast tumors. The ErbB-2 receptor encoded by this novel transcript harbors an in-frame deletion of 16 amino acids in the extracellular domain and can transform Rat-1 fibroblasts. Together, these observations argue that co-expression of ErbB-2 and ErbB-3 may play a critical role in the induction of human breast tumors, and raise the possibility that activating mutations in the ErbB-2 receptor may also contribute to this process.

Oncogenic activating mutations in the neu/erbB-2 oncogene are involved in the induction of mammary tumors.

Amplification and overexpression of erbB-2/neu is an important determinant in the initiation and progression of human breast cancer. Indeed, transgenic mice that over-express the neu proto-oncogene heritably develop mammary adenocarcinomas. Tumorigenesis in these transgenic strains is associated with activation of the intrinsic catalytic activity of Neu. In many of these tumors, activation of Neu occurs as a result of somatic mutations located within the transgene itself. Examination of the altered neu transcripts revealed the presence of in-frame deletions that encode aberrant Neu receptors lacking 5 to 12 amino acids within the extracellular domain proximal to the transmembrane region of Neu. In addition to these deletion mutants we have also detected single point mutations within this juxta-transmembrane region. The majority of the mutations analyzed affect the one of several conserved cysteine residues present within this region. Introduction of these activating mutations into the wild-type neu cDNA results in its oncogenic conversion. Taken together, these observations suggest that this cysteine-rich region plays an important role in regulating the catalytic activity of Neu.

Combination therapy with interleukin-2 and wild-type p53 expressed by adenoviral vectors potentiates tumor regression in a murine model of breast cancer.

Although cytokine gene transfer for cancer treatment can stimulate immune recognition and tumor regression in animal models, there is still a need for improvements to these strategies. In this study, we examined the efficacy of a combination gene therapy using adenovirus (Ad) 5 vectors expressing human interleukin-2 and the wild-type (wt) human p53 gene under control of the human cytomegalovirus immediate early promoter (AdIL-2 and Adp53wt, respectively). Infected murine cell lines and primary mouse tumor cells secreted high levels of IL-2 and over expressed the p53 protein for at least 9 days. After infection of cells with Adp53wt, DNA synthesis was significantly inhibited and apoptosis was induced within 3-5 days. Both vectors were tested in a transgenic mouse mammary adenocarcinoma model for antitumor response. Following a single intratumoral injection of mice bearing PyMT induced tumors, the combination of Adp53wt (1 x 10(9) pfu) plus a relatively low dose of AdIL-2 (1.5 x 10(8) pfu) caused regressions in 65% of the treated tumors without toxicity. Fifty percent of the treated mice remained tumor free and were immune to rechallenge with fresh tumor cells. In contrast, injection of either vector alone at this does resulted in only a delay in tumor growth. Only mice co-injected with Adp53wt and AdIL-2 showed specific antitumor cytolytic T lymphocyte (CTL) activity, indicating that the immune response involved in tumor regression was promoted by the combination therapy. These results suggest that cancer treatment strategies involving combined delivery of immunomodulatory and antiproliferative genes may be highly effective.