Later Stages Of Tumor Progression Research Articles

Correlation of malignancy parameters in colorectal carcinoma with up- and downstream signalling partners of STAT3

STAT3 (Signal Transducer and Activator of Transcription 3) is persistently activated in about 90% of colorectal carcinoma (CRC) cases. However, CRC cell lines show variability in their degree of constitutive and cytokine-inducible STAT3 tyrosine phosphorylation. We have employed both surgical biopsies from CRC patients and a large collection of permanent CRC cell lines to address (1), which tyrosine kinases are involved in aberrant STAT3 activation in CRC, and (2) in which ways dysregulated STAT3 activity contributes to malignancy-associated cell behavior in CRC. Western blot data obtained from analyzing CRC tumor specimens as well as vicinal normal tissue were quantified and statistically analysed. These studies revealed correlations between activated STAT3 on one hand and Src and JAK2 activity on the other (in particular in non-tumorous border tissue), and between Src and JAK1 activity (particularly in malignant tumor tissue). STAT3 activation shows a clear decrease in later stages of tumor progression. With regard to potential parameters of malignancy (here: invasiveness), we could show a striking coincidence of STAT3 activitation and strong expression of matrix metalloproteinases MMP-1 both by biochemical and histological analysis. For detailled studies, we chose from an extended panel of CRC cell lines as examples (1) HT-29, in which STAT3 activity is inducible by IL-6, and (2) C-10, in which a strong constitutive STAT3 phosphorylation is observed. In C-10 cells, blockade of STAT3 dimerization by an inhibitor peptide specifically led to cell death, whereas experimental activation of STAT3 in HT-29 cells via IL-6 resulted in enhanced cell growth, MMP-1 expression and invasiveness. In HT-29 cells, we investigated the role of IL-6-evoked STAT3 activity in the control of MMP-1 expression. Reporter gene experiments showed a direct influence of STAT3 activation on transcription from the human MMP-1 promoter. By analyzing both protein-protein and DNA-protein interactions within this promoter we obtained evidence for a complex mode a cooperative transcriptional regulation through STAT3 and AP-1. Additional STAT3 dimerization inhibitor compounds and CRC cell lines are currently being analyzed and the results will be discussed.

Effects of immunotherapy of IL-6 and IL-15 plasmids on transmissible venereal tumor in beagles

Canine transmissible venereal tumor (CTVT) is a tumor with low MHC antigen expression and is an ideal tumor model for studying the interactions between host immunity and cancer cells. CTVTs produce high concentrations of TGF-β to hamper the host immune responses and facilitate their growth progression. However, during the later stages of tumor progression, tumor-infiltrating lymphocytes secrete IL-6. This cytokine antagonizes TGF-β and restores the IFN-γ activities in promoting MHC antigen expression, and the NK cytotoxicity that has been repressed by TGF-β is also activated. In this study, we applied combinatory treatment of IL-6 plasmid and IL-15 plasmid ( pIL-6/pIL-15) to CTVT-bearing beagles. IL-6 was used as an anti-TGF-β cytokine; IL-15 was used to promote NK- and CTVT-specific cytotoxicity. After intratumoral pIL-6/pIL-15 delivery mediated by electroporation, MHC antigen expression on CTVT cells was dramatically increased from in less than 5.9% to up to 34% of the tumor cells. The proportion of CD8 + T cells infiltrating the tumor was also significantly elevated from 6.96 ± 0.23% to 21.63 ± 5.40%. In addition, the tumor-specific cytotoxicity was enhanced along with a marked increase in tumor-specific IFN-γ-producing cells. These immune responses are believed to be the important forces driving the tumor towards regression. The results indicate that pIL-6/pIL-15 combinatory immunotherapy may facilitate a promising and effective means of treating tumors.

Transforming growth factor-beta signaling and tumor angiogenesis

Transforming growth factor-beta (TGF-beta) family members are secreted multifunctional cytokines that play pivotal roles in development and disease. The prototypic member of this family, TGF-beta, plays a dual role in carcinogenesis, acting as a tumor suppressor in early stages and as tumor promoter in late stages of tumor progression. Numerous studies support the notion that pathological angiogenesis is one of the hallmarks of cancer. Tumor angiogenesis is regulated by a network of growth factors, including members of the TGF-beta family. TGF-beta acts in a context-dependent manner and can either stimulate or inhibit tumor angiogenesis. In this review, we discuss our current understanding on how TGF-beta family members affect endothelial and smooth muscle cell function and how perturbed TGF-beta signaling may contribute to tumor angiogenesis and tumor progression.

A novel Src kinase inhibitor reduces tumour formation in a skin carcinogenesis model

The Src family tyrosine kinases are key modulators of cancer cell invasion and metastasis and a number of Src kinase inhibitors are currently in clinical development for the treatment of solid tumours. However, there is growing evidence that Src is also upregulated at very early stages of epithelial cancer development. We have investigated the role of Src in mouse skin, which is one of the most tractable models of epithelial homoeostasis and tumorigenesis. We found that Src protein expression and activity was regulated during the normal hair cycle and was increased specifically during the proliferative anagen phase and also in response to the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). AZD0530, a selective Src inhibitor, prevented the TPA-induced proliferation of basal keratinocytes both in vivo and in vitro. Moreover, treatment with AZD0530 reduced papilloma formation following the well-established 7,12-dimethylbenz(a)anthracene/TPA skin carcinogenesis protocol but did not inhibit the subsequent proliferation of the papillomas. Furthermore, AZD0530 did not alter the malignant conversion of papillomas to squamous cell carcinoma suggesting a role for Src in early tumour development in the skin carcinogenesis model, rather than at later stages of tumour progression. Src expression and activity were also seen in human actinic keratoses that are hyperproliferative pre-malignant skin lesions, indicating that Src may also play a role in the early stages of human skin tumour development. Thus, Src inhibitors such as AZD0530 may therefore have chemopreventative properties in patients with hyperproliferative epidermal disorders.

TGFβ1 antagonistic peptides inhibit TGFβ1-dependent angiogenesis

The role of transforming growth factor beta (TGFβ) in tumor promotion and in angiogenesis is context-dependent. While TGFβ prevents tumor growth and angiogenesis in early phases of tumor development, evidence is accumulating about its pro-angiogenic and tumor promotion activities in late-stages of tumor progression. Here we have studied, in an experimental context previously reported to disclose the pro-angiogenic effects of TGFβ, the blocking activity of TGFβ antagonist peptides. In agreement with previous results, we have observed that TGFβ exerts a powerful pro-angiogenic activity on human normal dermal microvascular endothelial cells (MVEC), by promoting invasion and capillary morphogenesis in Matrigel. No apoptotic activity of TGFβ was observed. By RT-PCR we have shown that TGFβ up-regulates expression not only of plasminogen activator inhibitor type-1 (PAI-1), but also of the urokinase-type plasminogen activator receptor (uPAR), whose inhibition by specific antibodies blunted the TGFβ angiogenic response in vitro. The SMAD2/3 and FAK signaling pathways were activated by TGFβ in MVEC, as an early and late response, respectively. The use of two different TGFβ1 antagonist peptides, derived from TGFβ type III receptor sequence and 15-mer phage display technology, inhibited the signaling and pro-angiogenic response in vitro, as well as uPAR and PAI-1 up-regulation of MVEC following TGFβ challenge. The anti-angiogenic properties of both inhibitors were evident also in the in vivo TGFβ Matrigel Sponge Assay. These results may be relevant to develop a potentially fruitful strategy for the therapy of late-stage-associated tumor angiogenesis.

Keratin down-regulation in vimentin-positive cancer cells is reversible by vimentin RNA interference, which inhibits growth and motility

At later stages of tumor progression, epithelial carcinogenesis is associated with transition to a mesenchymal phenotype, which may contribute to the more aggressive properties of cancer cells and may be stimulated by growth factors such as epidermal growth factor and transforming growth factor-beta. Previously, we found that cells derived from a nodal metastatic squamous cell carcinoma are highly proliferative and motile in vitro and tumorigenic in vivo. In the current study, we have investigated the role of vimentin in proliferation and motility. Cells derived from nodal metastasis express high levels of vimentin, which is undetectable in tumor cells derived from a synchronous primary lesion of tongue. Vimentin expression was enhanced by epidermal growth factor and transforming growth factor-beta both independently and in combination. Use of RNA interference resulted in the generation of stable cell lines that express constitutively low levels of vimentin. RNA interference-mediated vimentin knockdown reduced cellular proliferation, migration, and invasion through a basement membrane substitute by 3-fold compared with nontargeting controls. In addition, cells with reduced vimentin reexpressed differentiation-specific keratins K13, K14, and K15 as a result of increased gene transcription as judged by quantitative PCR and promoter-reporter assays. Furthermore, cells in which vimentin expression was reduced showed a greatly decreased tumorigenic potential, as tumors developing from these cells were 70% smaller than those from control cells. The data suggest that reversal of the mesenchymal phenotype by inhibiting vimentin expression results in reexpression of epithelial characteristics and reduced tumor aggressiveness.

Mouse EP3 α, β, and γ Receptor Variants Reduce Tumor Cell Proliferation and Tumorigenesis in Vivo

Prostaglandin E(2), which exerts its functions by binding to four G protein-coupled receptors (EP1-4), is implicated in tumorigenesis. Among the four E-prostanoid (EP) receptors, EP3 is unique in that it exists as alternatively spliced variants, characterized by differences in the cytoplasmic C-terminal tail. Although three EP3 variants, alpha, beta, and gamma, have been described in mice, their functional significance in regulating tumorigenesis is unknown. In this study we provide evidence that expressing murine EP3 alpha, beta, and gamma receptor variants in tumor cells reduces to the same degree their tumorigenic potential in vivo. In addition, activation of each of the three mEP3 variants induces enhanced cell-cell contact and reduces cell proliferation in vitro in a Rho-dependent manner. Finally, we demonstrate that EP3-mediated RhoA activation requires the engagement of the heterotrimeric G protein G(12). Thus, our study provides strong evidence that selective activation of each of the three variants of the EP3 receptor suppresses tumor cell function by activating a G(12)-RhoA pathway.

Role of Bv8 in neutrophil-dependent angiogenesis in a transgenic model of cancer progression

The secreted Bv8 protein has been recently characterized as a regulator of myeloid cell mobilization and a neutrophil-derived mediator of tumor angiogenesis in several xenografts, but its role in tumor progression in an endogenous setting was unknown. The rat insulin promoter (RIP)-T-antigen (Tag) is a well characterized transgenic mouse model of multistage pancreatic beta-cell tumorigenesis. Also, the role of neutrophils in RIP-Tag angiogenic switching, as assessed by systemic ablation using anti-Gr1 antibodies at different stages of tumor progression, has been recently described. Here, we show that early treatment of RIP-Tag mice with anti-Bv8 antibodies resulted in a significant reduction in the number of angiogenic islets relative to control antibody-treated mice, implicating Bv8 in the angiogenic switch during neoplasia. Histological analysis showed a significant reduction in vascular surface areas in hyperplastic and angiogenic lesions in pancreatic islets from anti-Bv8-treated mice. Anti-Bv8 treatment also inhibited the mobilization and homing of CD11b+Gr1+ cells to the peripheral blood and the emerging neoplastic lesions. However, anti-Bv8 treatment had no effect on tumor vascularization or burden when initiated at later stages of tumor progression. The stage-dependent efficacy of anti-Bv8 treatment appears remarkably similar to that reported after neutrophil ablation, suggesting that Bv8 is an important mediator of neutrophil-dependent angiogenesis in this transgenic model. In summary, our studies verify a role for Bv8 in the mobilization and recruitment of myeloid cells and in the induction of tumor angiogenesis in the early stages of neoplastic progression.

The morphogenic function of E-cadherin-mediated adherens junctions in epithelial ovarian carcinoma formation and progression

E-cadherin expression is unusually regulated in epithelial ovarian carcinoma. It is not expressed in poorly cohesive ovarian surface epithelial (OSE) target cells, but is expressed in cohesive pre-malignant lesions and in highly cohesive, well-differentiated tumors where it is membrane associated, presumably in adherens junctions. E-cadherin expression is subsequently suppressed, or its function is disrupted, in late-stage invasive tumors. Here, we observed that increased E-cadherin expression in ovarian carcinoma cells was associated with increased E-cadherin promoter activity, increased adherens junction formation, decreased β-catenin signaling-dependent LEF-1 activity, and the generation of cohesive spheroids in basement membrane gel culture. Forced expression of wild-type E-cadherin in immortalized OSE cells initiated adherens junction formation, decreased LEF-1 activity, decreased the mesenchymal migration that is a characteristic of OSE cells that have been maintained in monolayer culture, and induced the formation of cohesive spheroids in basement membrane gels. Conversely, forced expression of a dominant-negative E-cadherin mutant in ovarian carcinoma cells disrupted adherens junctions, increased mesenchymal cell migration, and prevented spheroidal morphogenesis without altering LEF-1 signaling. Therefore, in addition to suppressing late-stage tumor progression, E-cadherin-mediated adherens junctions may also contribute to the initial emergence of a cohesive morphogenic phenotype that is a hallmark of differentiated epithelial ovarian carcinoma.

Modeling the VEGF–Bcl-2–CXCL8 Pathway in Intratumoral Agiogenesis

Recent experiments show that vascular endothelial growth factor (VEGF) is the crucial mediator of downstream events that ultimately lead to enhanced endothelial cell survival and increased vascular density within many tumors. The newly discovered pathway involves up-regulation of the anti-apoptotic protein Bcl-2, which in turn leads to increased production of interleukin-8 (CXCL8). The VEGF-Bcl-2-CXCL8 pathway suggests new targets for the development of anti-angiogenic strategies including short interfering RNA (siRNA) that silence the CXCL8 gene and small molecule inhibitors of Bcl-2. In this paper, we present and validate a mathematical model designed to predict the effect of the therapeutic blockage of VEGF, CXCL8, and Bcl-2 at different stages of tumor progression. In agreement with experimental observations, the model predicts that curtailing the production of CXCL8 early in development can result in a delay in tumor growth and vascular development; however, it has little effect when applied at late stages of tumor progression. Numerical simulations also show that blocking Bcl-2 up-regulation, either at early stages or after the tumor has fully developed, ensures that both microvascular and tumor cell density stabilize at low values representing growth control. These results provide insight into those aspects of the VEGF-Bcl-2-CXCL8 pathway, which independently and in combination, are crucial mediators of tumor growth and vascular development. Continued quantitative modeling in this direction may have profound implications for the development of novel therapies directed against specific proteins and chemokines to alter tumor progression.

Hypoxia/reoxygenation: A dynamic regulator of lysyl oxidase‐facilitated breast cancer migration

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.

Secondhand Suspicions: Breast Cancer and Passive Smoking

Does a young woman living with a smoker or taking a job working in a smoky bar have a greater chance of developing breast cancer? Some scientists believe that such situations can indeed raise a woman’s risk of developing breast cancer before the age of 50. Because epidemiological and toxicological studies show that women’s breast tissue may be especially sensitive to exposure to carcinogens prior to first pregnancy, these researchers contend that public education should be directed at alerting adolescents and young women to the potential risk. However, not everyone in the international public health community agrees that the evidence to date supports a link between passive smoking and breast cancer, and some say that women are being alarmed unnecessarily. This disagreement has sparked debate that is sometimes heated. The stakes are high because breast cancer is the most common cancer in women in industrialized countries, according to the WHO. It is the leading cancer killer of nonsmoking women, and second only to lung cancer deaths among women who smoke. Among the researchers interviewed for this article who disagree that there is enough evidence to link secondhand smoke (SHS) with breast cancer, the majority call the evidence to date “suggestive but not sufficient,” as the Surgeon General’s 2006 report, The Health Consequences of Involuntary Exposure to Tobacco Smoke, put it. That characterization is based largely on the fact that the research considered when the Surgeon General’s report was being amassed did not clearly link even active smoking to breast cancer. Researchers in this camp do, however, stress that ongoing campaigns to prohibit smoking in public will protect the whole of society against the wide variety of ills proven to be caused by SHS. These include lung cancer, cardiovascular disease, and sudden infant death syndrome, among others. A smaller group contends that the question of whether or not SHS causes breast cancer is a political issue with the potential to compromise the scientific process. “A premature decision about causality could jeopardize the credibility of the entire review process and all of the other, established effects of secondhand smoke,” says Michael Thun, national vice president of epidemiology and surveillance research for the American Cancer Society. Adds Valerie Beral, director of the University of Oxford Cancer Research UK Epidemiology Unit, “To prematurely come to conclusions about the causation when there is a big division in the scientific community . . . is bad science.” Thun debated the subject in a series of public forums held in conjunction with scientific meetings. Taking the opposing view was Kenneth C. Johnson, a research scientist with the Public Health Agency of Canada, who was one of the first scientists to discern a potential link. During the debates, Johnson pointed out there are about the same number of studies linking breast cancer to passive smoking as there were linking lung cancer to SHS in 1986, when the Surgeon General concluded that passive smoking caused lung cancer. Johnson also says that more of the breast cancer studies are statistically significant, and that the estimated risk for breast cancer is higher.

Alternative production of Bcl-2 and Bax by tumor cells determines the rates of in vivo tumor progression: Suggested mechanisms

The hypothesis tested in the study suggests that mechanisms of the earlier described delayed or accelerated tumor progression may be regulated by the antiapoptotic and proapoptotic cellular programs activated in stress reactions of transformed cells to the host normal cellular environment. Therefore, spontaneously transformed hamster cell line STHE, its bcl-2-transduced line STHE-Bcl-2, and 64 of their descendant tumor cell variants naturally selected in two in vivo regimes (local tumor growth versus dissemination) were examined. The role of Bcl-2 and the possible activation of endogenous death-signaling Bax, Ras, and HSP90/HSP70 stress proteins in STHE (Bcl-2+/-) tumor cell variants were studied in dynamics of in vivo tumor progression. The data demonstrate: (1) Immediate in vivo activation of Bax and of HSP90/HSP70 stress proteins in disseminated STHE cells on the background of accelerated tumor progression; (2) No immediate activation of Bax and the gradual downregulation of Bcl-2 in STHE-Bcl-2 cells on the background of delayed tumor progression; (3) Alternative and mutually suppressive character of Bcl-2 and Bax expression in both regimes of tumor progression; (4) In the later stages of tumor progression, the regular transit of the initial Bcl-2 antiapoptotic, Bax-suppressing program, and the delayed tumor progression towards Bcl-2 loss, activation of Bax, and acceleration of tumor progression. Thus, the delay of tumor progression is apparently determined by the ability of Bcl-2-expressing tumor cells to extinguish the cell-damaging environmental stress signals and Bax activation, while its acceleration correlates with Bcl-2 loss, activation of proapoptotic Bax, and tumor cells damage.

NSAID Activated Gene (NAG-1), a Modulator of Tumorigenesis

The NSAID activated gene (NAG-1), a member of the TGF-beta superfamily, is involved in tumor progression and development. The over-expression of NAG-1 in cancer cells results in growth arrest and increase in apoptosis, suggesting that NAG-1 has anti-tumorigenic activity. This conclusion is further supported by results of experiments with transgenic mice that ubiquitously express human NAG-1. These transgenic mice are resistant to the development of intestinal tumors following treatment with azoxymethane or by introduction of a mutant APC gene. In contrast, other data suggest a pro-tumorigenic role for NAG-1, for example, high expression of NAG-1 is frequently observed in tumors. NAG-1 may be like other members of the TGF-beta superfamily, acting as a tumor suppressor in the early stages, but acting pro-tumorigenic at the later stages of tumor progression. The expression of NAG-1 can be increased by treatment with drugs and chemicals documented to prevent tumor formation and development. Most notable is the increase in NAG-1 expression by the inhibitors of cyclooxygenases that prevent human colorectal cancer development. The regulation of NAG-1 is complex, but these agents act through either p53 or EGR-1 related pathways. In addition, an increase in NAG-1 is observed in inhibition of the AKT/GSK-3beta pathway, suggesting NAG-1 alters cell survival. Thus, NAG-1 expression is regulated by tumor suppressor pathways and appears to modulate tumor progression.

Evolution of VHL tumourigenesis in nerve root tissue

Haemangioblastomas are the key central nervous system manifestation of von Hippel-Lindau (VHL) disease, which is caused by germline mutation of the VHL gene. We have recently shown that 'tumour-free' spinal cord from patients with VHL disease contains microscopic, poorly differentiated cellular aggregates in nerve root tissue, which we descriptively designated 'mesenchymal tumourlets'. Here we have investigated spinal cord tissue affected by multiple tumours. We show that a small subset of mesenchymal tumourlets extends beyond the nerve root to form proliferative VHL-deficient mesenchyme and frank haemangioblastoma. We thus demonstrate that tumourlets present potential, but true precursor material for haemangioblastoma. We further show that intraradicular tumourlets consist of scattered VHL-deficient cells with activation of HIF-2alpha and HIF-dependent target proteins including CAIX and VEGF, and are associated with an extensive angiogenic response. In contrast, activation of HIF-1alpha was only observed in the later stages of tumour progression. In addition, ultrastructural examination reveals gradual transition from poorly differentiated VHL-deficient cells into vacuolated cells with a 'stromal' cell phenotype. The evolution of frank haemangioblastoma seems to involve multiple steps from a large pool of precursor lesions.

Transforming growth factor-β in cancer and metastasis

Transforming growth factor-beta (TGF-beta) is a multifunctional regulatory polypeptide that is the prototypical member of a large family of cytokines that controls many aspects of cellular function, including cellular proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis, immune surveillance, and survival. The actions of TGF-beta are dependent on several factors including cell type, growth conditions, and the presence of other polypeptide growth factors. One of the biological effects of TGF-beta is the inhibition of proliferation of most normal epithelial cells using an autocrine mechanism of action, and this suggests a tumor suppressor role for TGF-beta. Loss of autocrine TGF-beta activity and/or responsiveness to exogenous TGF-beta appears to provide some epithelial cells with a growth advantage leading to malignant progression. This suggests a pro-oncogenic role for TGF-beta in addition to its tumor suppressor role. During the early phase of epithelial tumorigenesis, TGF-beta inhibits primary tumor development and growth by inducing cell cycle arrest and apoptosis. In late stages of tumor progression when tumor cells become resistant to growth inhibition by TGF-beta due to inactivation of the TGF-beta signaling pathway or aberrant regulation of the cell cycle, the role of TGF-beta becomes one of tumor promotion. Resistance to TGF-beta-mediated inhibition of proliferation is frequently observed in multiple human cancers, as are various alterations in the complex TGF-beta signaling and cell cycle pathways. TGF-beta can exert effects on tumor and stromal cells as well as alter the responsiveness of tumor cells to TGF-beta to stimulate invasion, angiogenesis, and metastasis, and to inhibit immune surveillance. Because of the dual role of TGF-beta as a tumor suppressor and pro-oncogenic factor, members of the TGF-beta signaling pathway are being considered as predictive biomarkers for progressive tumorigenesis, as well as molecular targets for prevention and treatment of cancer and metastasis.

S043 Galanin Receptor Type 1 Inhibits Cell Proliferation in Squamous Carcinoma Cells via Erk1/2 Activation

allele can induce progression to SCC. Conclusions: These data support a gain-of-function role for p53R172H in oral SCC development and suggest that mutations in p53 not only contribute to late stages of tumor progression but also can cooperate with other genetic alterations in tumor initiation. We are currently analyzing expression profiles of p53R172H squamous cell papilloma in order to identify molecular mechanisms involved in mutant p53-mediated oral cancer progression.

S041 Identification of the Mechanism of 11q13 Amplification Leads to Detection of DNA Repair Defects in HNSCC Cell Lines

allele can induce progression to SCC. Conclusions: These data support a gain-of-function role for p53R172H in oral SCC development and suggest that mutations in p53 not only contribute to late stages of tumor progression but also can cooperate with other genetic alterations in tumor initiation. We are currently analyzing expression profiles of p53R172H squamous cell papilloma in order to identify molecular mechanisms involved in mutant p53-mediated oral cancer progression.

Osteopontin Knockdown Suppresses Tumorigenicity of Human Metastatic Breast Carcinoma, MDA-MB-435

Elevated expression of osteopontin (OPN), a secreted phosphoglycoprotein, is frequently associated with many transformed cell lines. Various studies suggest that OPN may contribute to tumor progression as well as metastasis in multiple tumor types. High levels of OPN have been reported in patients with metastatic cancers, including breast. We found that the expression of OPN corroborates with the aggressive phenotype of the breast cancer cells i.e. the expression of OPN is acquired as the breast cancer cells become more aggressive. To assess the role(s) of OPN in breast carcinoma, expression of endogenous OPN was knocked down in metastatic MDA-MB-435 human breast carcinoma cells using RNA interference. We targeted multiple regions of the OPN transcript for RNA interference, along with 'scrambled' and 'non-targeting siRNA pool' controls to distinguish between target-specific and potential off-target effects including interferon-response gene (PeIF2-alpha) induction. The OPN knockdown by shRNA suppressed tumor take in immunocompromised mice. The 'silenced' cells also showed significantly lower invasion and migration in modified Boyden chamber assays and reduced ability to grow in soft agar. Thus, in addition to the widely reported roles of OPN in late stages of tumor progression, these results provide functional evidence that OPN contributes to breast tumor growth as well.

Smad4 haploinsufficiency in mouse models for intestinal cancer

The Smad4(+/E6sad) mouse carries a null mutation in the endogenous Smad4 gene resulting in serrated adenomas and mixed polyposis of the upper gastrointestinal (GI) tract with 100% penetrance. Here, we show by loss of heterozygosity (LOH) analysis and immunohistochemistry (IHC) that, although the majority of the tumors appear at 9 months of age, somatic loss of the wild-type Smad4 allele occurs only at later stages of tumor progression. Hence, haploinsufficiency underlies Smad4-driven tumor initiation in the GI tract. As both the Apc and Smad4 tumor suppressor genes map to mouse chromosome 18, we have bred Smad4(+/E6sad) with the Apc(+/1638N) model to generate two distinct compound heterozygous lines carrying both mutations either in cis (CAS) or in trans (TAS). Strikingly, both models show increased tumor multiplicities when compared with the single mutant littermates, although CAS mice are more severely affected and became moribund at only 5-6 weeks of age. Phenotypic and molecular analyses indicate that Smad4 haploinsufficiency is sufficient to significantly affect tumor initiation and progression both prior to and upon loss of Apc function. Moreover, complete loss of Smad4 strongly enhances Apc-driven tumor formation.