Later Stages Of Tumor Progression Research Articles

APC/β-catenin-rich complexes at membrane protrusions regulate mammary tumor cell migration and mesenchymal morphology

BackgroundThe APC tumor suppressor is mutated or downregulated in many tumor types, and is prominently localized to punctate clusters at protrusion tips in migratory cells, such as in astrocytes where it has been implicated in directed cell motility. Although APC loss is considered an initiating event in colorectal cancer, for example, it is less clear what role APC plays in tumor cell motility and whether loss of APC might be an important promoter of tumor progression in addition to initiation.MethodsThe localization of APC and β-catenin was analyzed in multiple cell lines, including non-transformed epithelial lines treated with a proteasome inhibitor or TGFβ to induce an epithelial-to-mesenchymal transition (EMT), as well as several breast cancer lines, by immunofluorescence. APC expression was knocked down in 4T07 mammary tumor cells using lentiviral-mediated delivery of APC-specific short-hairpin (sh) RNAs, and assessed using quantitative (q) reverse-transcriptase (RT)-PCR and western blotting. Tumor cell motility was analyzed by performing wound-filling assays, and morphology via immunofluorescence (IF) and phase-contrast microscopy. Additionally, proliferation was measured using BrdU incorporation, and TCF reporter assays were performed to determine β-catenin/TCF-mediated transcriptional activity.ResultsAPC/β-catenin-rich complexes were observed at protrusion ends of migratory epithelial cells treated with a proteasome inhibitor or when EMT has been induced and in tumor cells with a mesenchymal, spindle-like morphology. 4T07 tumor cells with reduced APC levels were significantly less motile and had a more rounded morphology; yet, they did not differ significantly in proliferation or β-catenin/TCF transcriptional activity. Furthermore, we found that APC/β-catenin-rich complexes at protrusion ends were dependent upon an intact microtubule cytoskeleton.ConclusionsThese findings indicate that membrane protrusions with APC/β-catenin-containing puncta control the migratory potential and mesenchymal morphology of mammary tumor cells and suggest that APC loss during later stages of tumor progression might impact tumor cell dissemination or colonization.

Possible Risk Factors Associated with Radiation Proctitis or Radiation Cystitis in Patients with Cervical Carcinoma after Radiotherapy

Radiation proctitis and radiation cystitis are major complications for patients with cervical carcinoma following radiotherapy. In the present study, we aimed to determine the potential risk factors for the development of radiation proctitis and radiation cystitis after irradiation. A total of 1,518 patients with cervical carcinoma received external beam radiotherapy (EBRT) followed by high-dose-rate intracavitary brachytherapy (HDRICB) in our hospital. The incidences of radiation proctitis and radiation cystitis were recorded and associations with different factors (age, time period, tumor stage) were analyzed with χ2 (chi-squared) and Fisher exact tests. We found that 161 and 94 patients with cervical carcinoma were diagnosed with radiation proctitis and radiation cystitis, respectively, following radiotherapy. The prevalence of Grade I-II radiation proctitis or radiation cystitis was significantly lower than that of Grade III (radiation proctitis: 3.82% vs. 6.76%, P <0.05; radiation cystitis: 2.31% vs. 3.87%, P < 0.05) and was significantly enhanced in patients with late stage (IIIb) tumor progression compared to those in early stage (Ib, IIa) (P < 0.05). Moreover, the incidence of radiation proctitis and cystitis was not correlated with age or, time period following radiation, for each patient (P > 0.05). These observations indicate that a late stage of tumor progression is a potential risk factor for the incidence of radiation proctitis and cystitis in cervical carcinoma patients receiving radiotherapy.

TGF-β in Epithelial to Mesenchymal Transition and Metastasis of Liver Carcinoma

Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) represent the majority of hepatic malignancies and are among the most frequent causes of cancer deaths worldwide with a rising incidence in western countries. Upon progression of liver cancer, the epithelial to mesenchymal transition (EMT) is considered a key process that drives intrahepatic metastasis. EMT is the transformation of epithelial cells to a mesenchymal phenotype exacerbating motility and invasiveness of various epithelial cell types. In this review we focus on EMT in hepatic fibrosis, HCC and CCC that is governed by the transforming growth factor (TGF)-β signaling. This cytokine has been shown to play diverse and conflicting roles in malignant development, acting as a tumor-suppressor in early cancerogenesis but enhancing tumor dissemination in later stages of tumor progression. Importantly, TGF-β can induce EMT in a variety of cancers including HCC and CCC, even though the complex molecular mechanisms underlying this process are not yet fully understood. We aim at collecting recent findings on the impact of TGF-β-induced EMT in liver carcinoma progression and at discussing new insights on promising drugable targets for future therapeutic approaches against CCC and HCC.

Expression of mast cell proteases correlates with mast cell maturation and angiogenesis during tumor progression.

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression.

IRF8-dependent regulation of MMP3 influences tumor progression (162.1)

Abstract Matrix metalloproteinases (MMP) are involved in tissue remodeling under physiologic or pathologic conditions, including neoplasia. Although the stromelysin member, MMP3, has been implicated in early stages of tumorigenesis, its role during later stages of tumor progression are poorly understood. Thus, we tested the hypothesis that tumor-derived MMP3 influences the neoplastic process beyond the level of tumor initiation. We employed two mouse tumor models, CMS4 sarcoma and 4T1 mammary carcinoma, in which endogenous MMP3 levels could be experimentally altered. Using loss-of-function approaches, we showed that MMP3 downregulation reduced CMS4 or 4T1 tumor growth rate in vivo, but not in vitro. Moreover, MMP3 downregulation attenuated CMS4 experimental lung metastasis. Conversely using gain-of-function approaches, we found that MMP3 overexpression enhanced CMS4 tumor growth. MMP3 levels also directly correlated with invasion capacity in vitro, consistent with MMP mode of action. Lastly, we examined potential mechanisms of MMP3 regulation in our CMS4 model. Interestingly, we identified interferon regulatory factor-8 (IRF8), previously portrayed as a leukemic suppressor, as a negative regulator of MMP3 expression. Altogether, our data show that tumor-derived MMP3 impacts the neoplastic process at stages beyond initiation and may do so via novel IRF8-dependent mechanisms. Thus, these data unveil new factors of tumor progression, which may have implications for cancer therapy.

VALIDITY OF THYROID CANCER INCIDENCE DATA FOLLOWING THE CHERNOBYL ACCIDENT

The only clearly demonstrated cancer incidence increase that can be attributed to radiation from the Chernobyl accident is thyroid carcinoma in patients exposed during childhood or adolescence. Significant increases in thyroid disease were observed as soon as 4 y after the accident. The solid/follicular subtype of papillary carcinoma predominated in the early period after the accident. Morphological diagnosis of cancer in such cases, if no infiltrative growth is clearly visible, depends mainly on the nuclear criteria. Outdated equipment and insufficient quality of histological specimens impeded reliable evaluation of the nuclear criteria. Access to foreign professional literature has always been limited in the former Soviet Union. The great number of advanced tumors observed shortly after the accident can be explained by the screening effect (detection of previously neglected cancers) and by the fact that many patients were brought from non-contaminated areas and registered as Chernobyl victims. It is also worth noting that exaggeration of the Chernobyl cancer statistics facilitated the writing of dissertations, financing of research, and assistance from outside the former Soviet Union. "Chernobyl hysteria" impeded nuclear energy production in some countries, thus contributing to higher prices for fossil fuel. The concluding point is that since post-Chernobyl cancers tend on average to be in a later stage of tumor progression, some published data on molecular or immunohistochemical characteristics of Chernobyl-related cancers require reevaluation.

Matrix metalloproteinases in tumorigenesis: an evolving paradigm.

Proteases are crucial for development, tissue remodeling, and tumorigenesis. Matrix metalloproteinases (MMPs) family, in particular, consists of more than 20 members with unique substrates and diverse function. The expression and activity of MMPs in a variety of human cancers have been intensively studied. MMPs have well-recognized roles in the late stage of tumor progression, invasion, and metastasis. However, increasing evidence demonstrates that MMPs are involved earlier in tumorigenesis, e.g., in malignant transformation, angiogenesis, and tumor growth both at the primary and metastatic sites. Recent studies also suggest that MMPs play complex roles in tumor progression. While most MMPs promote tumor progression, some of them may protect the host against tumorigenesis in a context-dependent manner. MMPs have been chosen as promising targets for cancer therapy on the basis of their aberrant up-regulation in malignant tumors and their ability to promote cancer metastasis. Although preclinical studies testing the efficacy of MMP suppression in tumor models were so encouraging, the results of clinical trials in cancer patients have been rather disappointing. Here, we review the complex roles of MMPs and their endogenous inhibitors such as tissue inhibitors of metalloproteinase in tumorigenesis and strategies in suppressing MMPs.

Abstract 1452: The estrogen receptor β and its splice variant estrogen receptor β2 inhibits expression of Runx2, a key regulator of bone metastasis while only estrogen receptor β inhibits proliferation of the prostate cancer cell line PC3

Abstract Prostate cancer often metastases to bone in its later stages of tumor progression. We wanted to investigate if estrogen receptor β (ERβ) and/or its splice variant estrogen receptor β2 (ERβ2) are affecting signaling pathways involved in bone metastasis of prostate cancer. ERβ is progressively lost during prostate cancer progression while the splice variant ERβ2 is still expressed in advanced prostate cancer. ERβ2 is a 495 amino acid long splice variant of estrogen receptor ERβ. Using prostate cancer cell line, PC3 stably expressing ERβ or ERβ2, we found that both receptors inhibit migration in wound healing assays and are furthermore, repressing the expression of the bone metastasis regulator Runx2 and its target genes Slug and MMP9. In addition, we found by assaying the TCF/LEF reporter (TopGlow), that ERβ represses β-catenin signaling and it is also known that Runx2's expression is dependent on β-catenin activity. This gives a possible mechanism, by which ERβ represses Runx2 expression, is by inhibiting β-catenin signaling. This opens up a new pathway for the regulation of Runx2 by ERβ. On the other hand, we found that only ERβ and not ERβ2 inhibits proliferation and expression of the proliferation marker Skp2, which we have previously found to be a target gene of ERβ. The lack of antiproliferative activity of ERβ2 suggest that this splice variant could still be expressed in highly proliferative prostate cancer while at the same time inhibiting bone metastasis. In conclusion we found that both ERβ and ERβ2 are good targets for treatment of prostate cancer that might metastasize to the bone. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1452. doi:10.1158/1538-7445.AM2011-1452

Abstract 2841: Inducible modulation of NF-kappaB in macrophages reveals that timing of intervention may be critical to therapeutic outcome during mammary tumorigenesis

Abstract Elevated levels of NF-kappaB signaling are associated with multiple cancers. Therefore, it is not surprising that inhibitors of NF-kappaB are being investigated as potential therapeutics. While extensive efforts are being made to identify inhibitors there is less focus on investigating the effects of inhibition in specific cell types and during defined stages of tumor progression. We have developed murine transgenic models that enable us to induce expression of an activator or inhibitor of NF-kappaB in macrophages, by adding doxycycline to the drinking water of mice. We have combined these with the Polyoma model of mammary cancer to investigate the effects of modulation of NF-kappaB signaling specifically within macrophages. Given several recent publications we were predicting that activation of NF-kappaB would have pro-tumor effects. However, using a tail vein injection model, our data identifies a stage during progression in which activation of NF-kappaB in macrophages significantly limits metastasis. Activation of NF-kappaB alters immune cell populations and results in rapid killing of tumor cells during the seeding phase. In this model inhibition of NF-kappaB in macrophages during this critical stage of metastasis is pro-tumor. This suggests that while inhibition of NF-kappaB at later stages of tumor progression may have therapeutic benefit, inhibition during the seeding phase my lead to the opposite effect. Our studies highlight the importance of continued investigation into aspects of cell specificity and timing that may change the potential outcome of a clinical trial using NF-kappaB inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2011-2841

Association of pituitary tumor transforming gene expression with early oral tumorigenesis and malignant progression of precancerous lesions

Pituitary tumor transforming gene (PTTG1) is overexpressed in many types of human cancers and is involved in late-stage tumor progression. The role of PTTG1 in initiating tumorigenesis is unclear. PTTG1 expression was assessed in precancerous lesions and squamous cell carcinomas of the oral cavity (OSCC). The association between the protein expression and clinicopathologic parameters was analyzed. The expression level of PTTG1 upon carcinogen treatment was also investigated. PTTG1 was overexpressed in both precancerous lesions and OSCC. The expression of PTTG1 was associated with carcinogen exposure in vivo and in vitro. PTTG1 overexpression was an independent factor for oral cancer development in precancerous lesions. This study provides the first evidence that PTTG1 is involved in the early stages of oral tumorigenesis. Carcinogen exposure may cause the initial induction of PTTG1 expression in oral precancerous lesions. PTTG1 overexpression is a potential prognosticator for malignant progression of oral precancerous lesions.

CIITA‐driven MHC‐II positive tumor cells: Preventive vaccines and superior generators of antitumor CD4+ T lymphocytes for immunotherapy

In our study, we have investigated whether tumors of distinct histological origin can be rejected if expressing CIITA-driven MHC class II molecules. Moreover, we assessed whether antitumor lymphocytes generated by this approach could be used as an immunotherapeutic tool for established cancers. Stable CIITA-transfectants of C51colon adenocarcinoma, RENCA renal adenocarcinoma, WEHI-164 sarcoma as well as TS/A mammary adenocarcinoma were generated. Tumor cells transfectants were injected in vivo, and their growth kinetics and recipient's immune response were analyzed. Tumor rejection and/or retardation of growth was found for the first 3 CIITA-transfected tumor cell lines and confirmed for TS/A-CIITA. Animals rejecting CIITA-transfected tumors acquired specific immunological memory as demonstrated by resistance to challenge with parental tumors. Adoptive cell transfer experiments demonstrated that tumor immunity correlates with the efficient priming of CD4(+) T helper cells and the consequent activation of CD8(+) T lymphocytes. T cells from TS/A-vaccinated mice were used in an adoptive immunotherapy model of established tumors. The results showed the cure at early stages and significantly prolonged survival at later stages of tumor progression. Importantly, CD4(+) T cells were clearly superior to CD8(+) T cells in antitumor protective function. Interestingly, the protective phenotype was associated to both a Th1 and Th2 polarization of the immune effectors. These results establish the general application of our tumor vaccine model and disclose the additional application of this strategy for producing better lymphocyte effectors for adoptive antitumor immunotherapy.

Stromelysin-1 (MMP-3) is a target and a regulator of Wnt1-induced epithelial-mesenchymal transition (EMT)

Matrix metalloproteinases (MMPs) play a well-defined role in later stages of tumor progression. However, there has been evidence that they also contribute to earlier stages of malignant transformation. The Wnt signaling transduction pathway plays a critical role in development and in the pathogenesis of many epithelial cancers. Here we have used Wnt1-induced epithelial-mesenchymal transition (EMT) in C57MG murine mammary epithelial cells to study the role of MMPs in this early step of malignant progression. Overexpression of Wnt1 in C57MG cells promoted EMT, the translocation of b-catenin from the cell membrane to the nucleus and its transcriptional activity, cell proliferation and cell motility. Simultaneously, we observed an increased expression of stromelysin-1 (MMP-3) and a 5.5-fold increase in MMP-3 promoter activity in C57MG cells expressing Wnt1 compared with C57MG cells. Treatment of Wnt-overexpressing cells with MMP inhibitor AG3340 decreased MMP-3 expression. We also found evidence that MMP-3 and Wnt3a cooperate in enhancing the transcriptional activity of β-catenin in C57MG cells. Consistently, the effects of Wnt1 on EMT, proliferation, and migration were inhibited by MMP inhibitors, or upon downregulation of MMP-3 by siRNA. These results suggest that MMP-3 is both a direct transcriptional target and a necessary contributor of the Wnt/b-catenin signaling pathway.

Overexpression of the Pituitary Tumor Transforming Gene Induces p53-dependent Senescence through Activating DNA Damage Response Pathway in Normal Human Fibroblasts

Pituitary tumor transforming gene (PTTG1, securin) is involved in cell-cycle control through inhibition of sister-chromatid separation. Elevated levels of PTTG1 were found to be associated with many different tumor types that might be involved in late stage tumor progression. However, the role of PTTG1 in early stage of tumorigenesis is unclear. Here we utilized the adenovirus expression system to deliver PTTG1 into normal human fibroblasts to evaluate the role of PTTG1 in tumorigenesis. Expressing PTTG1 in normal human fibroblasts inhibited cell proliferation. Several senescence-associated (SA) phenotypes including increased SA-beta-galactosidase activities, decreased bromodeoxyuridine incorporation, and increased SA-heterochromatin foci formation were also observed in PTTG1-expressing cells, indicating that PTTG1 overexpression induced a senescent phenotype in normal cells. Significantly, the PTTG1-induced senescence is p53-dependent and telomerase-independent, which is distinctively different from that of replicative senescence. The mechanism of PTTG1-induced senescence was also analyzed. Consistent with its role in regulating sister-chromatid separation, overexpression of PTTG1 inhibited the activation of separase. Consequently, the numbers of cells with abnormal nuclei morphologies and chromosome separations were increased, which resulted in activation of the DNA damage response. Thus, we concluded that PTTG1 overexpression in normal human fibroblasts caused chromosome instability, which subsequently induced p53-dependent senescence through activation of DNA-damage response pathway.

Abstract 5307: Indomethacin reverts tumor mediated immune suppression in a murine lung adenocarcinoma

Abstract Tumor-induced immune suppression is widespread in patients and experimental animals with malignant tumors and is likely to be a significant impediment to immunotherapy. Multiple mechanisms are thought to facilitate this suppression state, being Myeloid-Derived Suppressor Cells (MDSC) and T regulatory cells (Tregs) major contributors. Our aim was to analyze immune parameters indicative of immune suppression in BALB/C mice bearing LP07 (COX+) lung tumor (TBM) during tumor growth and the effect of a Non Steroidal Anti-inflammatory drug (NSAID), Indomethacin, which is a non-specific COX inhibitor, on these parameters. We determined 1) the effect of Indomethacin on LP07 tumor growth; 2) the content of MDSC and Treg cells in lymphoid organs (spleen, tumor-draining lymph nodes-TDLN-) and in the tumors at different times by FACS; 3) arginase activity in lung, spleen, liver and tumor measured as µg Urea/mg protein; 4) specific Delayed Type Hypersensitivity (DTH) response with formalinized LP07 cells, by the foot pad swelling assay. Results: Indomethacin treatment significantly inhibited tumor growth. The percentage of MDSC (CD11b+ Gr1+) augmented significantly in the spleen of TBM in advanced stages of tumor progression (33 days): Normal: 2,6%; TBM 10 days: 2,4%; TBM 20 days: 5,6%; TBM 33 days: 8,3%, p&amp;lt;0,05 vs control. This rise was prevented by Indomethacin treatment (4,6%). Intratumoral and TDLN CD4+CD25+Foxp3+ Tregs frequency augmented during tumor growth, peaking at day 20. Interestingly, Indomethacin treatment decreased Treg cell and MDSC induction (p&amp;lt;0.05) in TDLN. Arginase activity increased in all analyzed tissues of TBM compared to those of control mice. Furthermore arginase activity increase in the spleen was in line with MDSC induction. Specific DTH response decreased in late stages of tumor progression, while Indomethacin treatment reversed this inhibition (TBM: 0,06 ± 0,045 mm; Indo:0,14 ± 0,038 mm). Conclusion: In our model, the increment in MDSC and Tregs frequency during tumor progression correlates with specific immunesuppression demonstrated by DTH responses and increased arginase activity. We suggest that Indomethacin treatment prevented this immunesuppression by modulating COX activity in the tumor microenvironment, which is known to activate MDSC and differentiate Treg cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5307.

Abstract 238: NPAS3 is a novel late-stage acting progression factor in gliomas with tumor-suppressive functions

Abstract Background: Primary brain tumors are among the top 5 causes of cancer-related deaths, with astrocytomas being the most common. Despite current therapies, patients with Glioblastoma multiforme (GBMs) (the most common sub-type of gliomas) unfortunately still succumb to a median survival of ∼ 1 year. In our effort to better comprehend the genetic basis of gliomas, we explored the prospects for new therapeutic targets. We previously cloned and characterized the function of NPAS3, a transcription factor which maps to human chromosome 14. Our principal aim is to comprehend the disease associations of NPAS3, since we recently identified expression in human astrocytes. We investigated NPAS3 as a candidate for astrocytomas based on findings archived from the Cancer Genome Project demonstrating a loss of NPAS3 expression and with loss-of-function deletions of human chromosome 14 with NPAS3 in 30-50% of astrocytomas. Methods-Results: After undertaking extensive functional analyses of NPAS3 using human surgical astrocytoma specimens, glioma cell line and human astrocyte cell line models, we now have novel and strong evidence supporting NPAS3 as an astrocytoma tumor suppressor involved in late-stage tumor progression. Our data in support of this discovery are based on: 1) Absent NPAS3 expression is predominant in high grade astrocytomas (79-83%), in comparison to low grade astrocytomas (29-35%) suggestive of an expression pattern typical of tumor suppressive- late stage progression factors. 2) Loss of function mutations (large deletions, frameshift, missense) of NPAS3, which are associated with a loss of heterozygosity of the NPAS3 locus are identified in GBMs. 3) Absent NPAS3 expression is predominant in &amp;gt;60% of malignant human glioma cell lines. 4) An over-expressed NPAS3 in malignant glioma cell lines suppresses the transformation potential, while the converse reduced expression promotes an increase in transformation potential. 5) A reduced NPAS3 expression (efficiency &amp;gt;90%) in concert with the human papillomavirus E6/E7 oncogenes, transforms a well characterized TERT immortalized human astrocyte cell line and promotes the growth of anaplastic astrocytomas, while an over-expressed NPAS3 suppresses the transformation. 6) NPAS3 mediates its tumor suppressive progression factor role in our glioma cell line and human astrocyte models by the control of cell cycle, proliferation and apoptosis. Conclusions: Our data provide compelling findings of NPAS3 as a novel gene involved in the cause of astrocytomas, with tumor suppressive and late-stage acting progression factor roles. We believe that this body of work is highly significant in our quest to better understand the biology of astrocytomas, and with prospects for improved targeted therapies in the treatment of this currently and relatively incurable disease. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 238.

Immune Promotion of Epithelial-mesenchymal Transition and Generation of Breast Cancer Stem Cells

Elements of the immune system act as intimate regulators of cancer progression, inhibiting early stages of tumor growth, through immunosurveillance while facilitating later stages of tumor progression. Recent findings have revealed that activated CD8 T cells can stimulate mammary epithelial tumor cells to undergo epithelial-mesenchymal transition (EMT) and to acquire the greatly increased tumorigenic capability and chemotherapeutic resistance of breast cancer stem cells (BCSC). These studies provide a window to understanding how BCSC arise and are maintained within tumors, and how to best target these processes for therapeutic benefit.

Tumor-promoting role of signal transducer and activator of transcription (Stat)1 in late-stage melanoma growth

A large-scale gene expression study of melanoma metastases was performed to identify genes involved in late-stage tumor progression. Overall 248 genes, out of more than 47,000 tested, are differentially expressed when comparing peripheral areas (invasion front) with central tumor areas of melanoma metastases. As determined by gene ontology analysis, members of the STAT signaling pathway show significant enrichment. In particular, Stat1 is highly expressed in peripheral compared with central tumor areas. In line with this, stable knockdown of STAT1 in metastatic melanoma cells significantly impairs their migratory and invasive capacity in wounding and matrigel assays. Moreover, STAT1 knockdown affects the metastatic behavior of melanoma cells in a mouse model of melanoma metastasis. Taken together, these data suggest that Stat1 might play a role in late-stage melanoma progression. Interference with the Stat1 pathway could have therapeutic implications for late-stage melanoma patients.

Hypoxia-activated Smad3-specific Dephosphorylation by PP2A

The transforming growth factor-beta (TGF-beta) maintains epithelial homeostasis and suppresses early tumor formation, but paradoxically at later stages of tumor progression, TGF-beta promotes malignancy. TGF-beta activates phosphorylation of Smad2 and -3 effectors. Smad2 and -3 are known to have different functions, but differential regulation of their phosphorylation has not been described. Here we show that upon hypoxia, the TGF-beta-induced phosphorylation of Smad3 was inhibited, although Smad2 remained phosphorylated. The inhibition of Smad3 phosphorylation was not due to TGF-beta receptor inactivation. We show that Smad3 was dephosphorylated by PP2A (protein phosphatase 2A) specifically under hypoxic conditions. The hypoxic Smad3 dephosphorylation required intact expression of the essential scaffold component PR65 of PP2A. PP2A physically interacted with Smad3 that occurred only in hypoxia. Accordingly, Smad3-associated PP2A activity was found under hypoxic conditions. Hypoxia attenuated the nuclear accumulation of TGF-beta-induced Smad3 but did not affect Smad2. Moreover, the influence of TGF-beta on a set of Smad3-activated genes was attenuated by hypoxia, and this was reversed by chemical PP2A inhibition. Our data demonstrate the existence of a Smad3-specific phosphatase and identify a novel role for PP2A. Moreover, our data implicate a novel mechanism by which hypoxia regulates growth factor responses.

TGF-β1–induced expression of human Mdm2 correlates with late-stage metastatic breast cancer

The E3 ubiquitin ligase human murine double minute (HDM2) is overexpressed in 40%-80% of late-stage metastatic cancers in the absence of gene amplification. Hdm2 regulates p53 stability via ubiquitination and has also been implicated in altering the sensitivity of cells to TGF-beta1. Whether TGF-beta1 signaling induces Hdm2 expression leading to HDM2-mediated destabilization of p53 has not been investigated. In this study, we report that TGF-beta1-activated SMA- and MAD3 (Smad3/4) transcription factors specifically bound to the second promoter region of HDM2, leading to increased HDM2 protein expression and destabilization of p53 in human cancer cell lines. Additionally, TGF-beta1 expression led to Smad3 activation and murine double minute 2 (Mdm2) expression in murine mammary epithelial cells during epithelial-to-mesenchymal transition (EMT). Furthermore, histological analyses of human breast cancer samples demonstrated that approximately 65% of late-stage carcinomas were positive for activated Smad3 and HDM2, indicating a strong correlation between TGF-beta1-mediated induction of HDM2 and late-stage tumor progression. Identification of Hdm2 as a downstream target of TGF-beta1 represents a critical prosurvival mechanism in cancer progression and provides another point for therapeutic intervention in late-stage cancer.

Evaluation of malignancy-risk gene signature in breast cancer patients

We recently developed a malignancy-risk gene signature that was shown to identify histologically-normal tissues with a cancer-like profile. Because the signature was rich with proliferative genes, we postulated it might also be prognostic for existing breast cancers. We evaluated the malignancy risk gene signature to see its clinical association with cancer relapse/progression, and cancer prognosis using six independent external datasets. Six independent external breast cancer datasets were collected and analyzed using the malignancy risk gene signature designed to assess normal breast tissues. Evaluation of the signature in external datasets suggested a strong clinical association with cancer relapse/progression, and prognosis with minimal overlap of signature gene sets. These results suggest a prognostic role for the malignancy risk gene signature in the assessment of existing cancer. Proliferative biology dominates not only the earliest stages of tumor development but also later stages of tumor progression and metastasis.