Agnes Witkiewicz

RB-pathway disruption in breast cancer: differential association with disease subtypes, disease-specific prognosis and therapeutic response.

In breast cancer, inactivation of the RB tumor suppressor gene is believed to occur via multiple mechanisms to facilitate tumorigenesis. However, the prognostic and predictive value of RB status in disease-specific clinical outcomes has remained uncertain. We investigated RB pathway deregulation in the context of both ER-positive and ER-negative disease using combined microarray datasets encompassing over 900 breast cancer patient samples. Disease-specific characteristics of RB pathway deregulation were investigated in this dataset by evaluating correlation among pathway genes as well as differential expression across patient tumor populations defined by ER status. Survival analysis among these breast cancer samples demonstrates that the RB-loss signature is associated with poor disease outcome within several independent cohorts. Within the ER-negative subpopulation, the RB-loss signature is associated with improved response to chemotherapy and longer relapse-free survival. Additionally, while individual genes in the RB target signature closely reproduce its prognostic value, they also serve to predict and monitor response to therapeutic compounds, such as the cytostatic agent PD-0332991. These results indicate that the RB-loss signature expression is associated with poor outcome in breast cancer, but predicts improved response to chemotherapy based on data in ER-negative populations. While the RB-loss signature, as a whole, demonstrates prognostic and predictive utility, a small subset of markers could be sufficient to stratify patients based on RB function and inform the selection of appropriate therapeutic regimens.

Expression of indoleamine 2,3-dioxygenase in metastatic pancreatic ductal adenocarcinoma recruits regulatory T cells to avoid immune detection.

BACKGROUND The mechanism by which pancreatic ductal adenocarcinoma (PDA) cells escape immune detection and survive in lymph nodes is poorly understood. One possible mechanism by which PDA cells can escape immune detection is through upregulation of indoleamine 2,3-dioxygenase (IDO), an enzyme that can starve T lymphocytes of tryptophan. STUDY DESIGN Seventeen cases of PDA were evaluated by immunohistochemistry for expression of IDO in tumor cells and the number of Forkhead box p3-expressing regulatory T cells. To validate IDO protein expression, Western blot analysis for IDO was performed on primary pancreatic cancer cell-line protein lysates. RESULTS Upregulation of IDO in metastatic PDA cells was associated with an increased number of regulatory T cells. Cytoplasmic IDO expression was present in all tumors (primary and metastatic) from patients with lymph node metastases. Intensity of staining was stronger in the corresponding metastatic foci when compared with the primary tumor. Three nonmetastatic PDAs were negative or only focally positive for IDO. Additionally, IDO expression in PDA was independent of tumor histologic grade. Forkhead box p3 regulatory T cells were increased in lymph nodes containing metastatic tumor cells expressing IDO. Using Western blot and reverse transcriptase polymerase chain reaction analysis, we validated that IDO expression in pancreatic cancer cells is induced by interferon-gamma as reported previously. CONCLUSIONS These data support the notion that metastatic PDA cells select for overexpression of IDO to evade immunologic detection. Future studies will define whether IDO expression in PDA patients with lymph node-positive metastases correlates with decreased survival. In addition, inhibition of IDO in PDA patients can be useful to enhance immunotherapeutic strategies.

A Western-Type Diet Accelerates Tumor Progression in an Autochthonous Mouse Model of Prostate Cancer

Epidemiological studies have provided evidence suggesting an important role for diet and obesity in the development of cancer. Specifically, lipid nutrients of the diet have been identified as important regulators of tumor development and progression. In the present study, we have examined the role of dietary fat and cholesterol in the initiation and progression of prostate cancer using the well-characterized TRAMP mouse model. Consumption of a Western-type diet--that is, enriched in both fat and cholesterol--accelerated prostate tumor incidence and tumor burden compared to mice fed a control chow diet. Furthermore, we also show that this diet increased the extent and the histological grade of prostate tumors. These findings were confirmed by the presence of increased levels of protein markers of advanced tumors in prostates obtained from animals fed a Western-type diet compared to those obtained from control animals. Increased lung metastases in animals fed a Western-type diet were also observed. In addition, we found that with a Western diet, animals bearing tumors presented with reduced plasma cholesterol levels compared with animals fed a control diet. Finally, we show that tumors obtained from animals fed a Western-type diet displayed increased expression of the high-density lipoprotein receptor SR-BI and increased angiogenesis. Taken together, our data suggest that dietary fat and cholesterol play an important role in the development of prostate cancer.

Cytoplasmic accumulation of the RNA binding protein HuR is central to tamoxifen resistance in estrogen receptor positive breast cancer cells

With prolonged exposure, a majority of estrogen receptor positive cancers develop resistance to tamoxifen and subsequent therapies including selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs). While much is known about overexpression of key growth promoting receptors including EGF, erbB2/Her2 and IGF receptors and subsequent activation of MAPK signaling associated with resistance, the underlying mechanism in the development of resistance still remains unknown. We found that inhibition of JNK, a member of the MAPK family, decreases cytoplasmic accumulation of the RNA binding protein HuR. This data combined with previous reports that erbB2/Her2 and IGF-IR signals through JNK, led us to hypothesize that cytoplasmic accumulation of HuR may be a key contributor to development of tamoxifen resistance. Therefore, we tested the effect of HuR expression on tamoxifen responsiveness in both tamoxifen sensitive MCF7 and tamoxifen resistant BT474 cell lines. We found that decreasing the cytoplasmic HuR levels in the cells increases tamoxifen responsiveness in both cell lines. Conversely, the overexpression of HuR establishes tamoxifen resistance in MCF7 cells. Therefore, our data indicate that HuR is central to tamoxifen resistance. Interestingly, we found that acute exposure (24 and 48 h) of MCF7 cells to tamoxifen increased cytoplasmic levels of HuR and concomitantly it’s ligand pp32, suggesting a novel molecular mechanism of resistance and acute response to tamoxifen through increased stability of mRNA transcripts that code for drug-resistant transcripts. Indeed, evaluation of primary breast tumors revealed a correlation between tumor grade, tamoxifen responsiveness and cytoplasmic HuR status. Therefore, inhibition of the cytoplasmic accumulation of HuR concomitantly with the administration of current therapeutics may be a successful treatment strategy. Our data describe a novel mechanism for the development of tamoxifen resistance and is the first study to identify an RNA binding protein as a key mediator of resistance in breast cancer cells

Angiotensin II induces vascular endothelial growth factor in pancreatic cancer cells through an angiotensin II type 1 receptor and ERK1/2 signaling.

Vascular endothelial growth factor (VEGF) is a crucial pro-angiogenic component in pancreatic ductal adenocarcinoma (PDA), and its high expression levels have been correlated with poor prognosis and early postoperative recurrence. We have recently shown that high levels of angiotensin II (AngII) type 1 receptor (AT1R) correlate and colocalize with VEGF in invasive PDA and that AngII induces VEGF expression in PDA cell lines. In this study, we explored the signaling mechanisms involved in the AngII-mediated VEGF induction and correlated AT1R and VEGF expression in noninvasive precursor lesions. An AT1R antagonist significantly (p < 0.05) inhibited the AngII-mediated induction of VEGF messenger RNA and protein in all PDA cell lines. AngII-VEGF induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a mitogen-activated protein kinase signaling mechanism. AngII activated the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), but not p38 or c-Jun NH2-terminal MAP kinases. Inhibition of ERK1/2 activation reduced the AngII-induced VEGF synthesis. Immunohistochemical analysis of precursor lesions showed increased expression of AT1R in most ductal cells undergoing metaplasia. Pancreatic intraepithelial neoplasms showed more intense AT1R staining when compared to intraductal papillary mucinous neoplasms, which showed heterogeneous immunoreactivity. VEGF followed the same distribution pattern of AT1R in both lesions. AT1R expression in the premalignant pancreatic lesions suggests its involvement in tumor progression and angiogenesis. Our mechanistic findings provide the first insight into an AngII-initiated signaling pathway that regulates PDA angiogenesis. An AT1R-mediated VEGF induction suggests the possibility of AT1R blockade as a novel therapeutic strategy to control angiogenesis in PDA.

The Cell Fate Determination Factor Dachshund Inhibits Androgen Receptor Signaling and Prostate Cancer Cellular Growth

Initially isolated as the dominant suppressor of the mutant epidermal growth factor receptor (ellipse), the Dachshund gene plays a key role in metazoan development regulating the Retinal Determination Gene Network. Herein, the DACH1 gene was expressed in normal prostate epithelial cells with reduced expression in human prostate cancer. DACH1 inhibited prostate cancer cellular DNA synthesis, growth in colony forming assays, and blocked contact-independent growth in soft agar assays. DACH1 inhibited androgen receptor (AR) activity, requiring a conserved DS Domain (Dachshund domain conserved with Ski/Sno) that bound NCoR/HDAC and was recruited to an androgen-responsive gene promoter. DACH1 inhibited ligand-dependent activity of AR mutations identified in patients with androgen-insensitive prostate cancer. The DS domain was sufficient for repression of the AR wild-type but failed to repress an AR acetylation site point mutant. These studies show a role for the Retinal Determination Gene Network in regulating cellular growth and signaling in prostate cancer.

Gangliocytic paraganglioma: case report and review of the literature

Gangliocytic paraganglioma is a rare tumor, which occurs nearly exclusively in the second portion of the duodenum. Generally, this tumor has a benign clinical course, although rarely, it may recur or metastasize to regional lymph nodes. Only one case with distant metastasis has been reported. We present a case of duodenal gangliocytic paraganglioma treated first by local resection followed by pylorus-preserving pancreaticoduodenectomy. Examination of the first specimen revealed focal nuclear pleomorphism and mitotic activity, in addition to the presence of three characteristic histologic components: epithelioid, ganglion, and spindle cell. In the subsequent pancreaticoduodenectomy specimen, there was no residual tumor identified in the periampullary area, but metastatic gangliocytic paraganglioma was present in two of seven lymph nodes. This case report confirms the malignant potential of this tumor. We review the published literature on gangliocytic paragangliomas pursuing a malignant course. We conclude that surgical therapy of these neoplasms should not be limited to local resection, as disease recurrence, lymph node involvement, and rarely distant metastasis may occur.

Evaluating the drug-target relationship between thymidylate synthase expression and tumor response to 5-fluorouracil. Is it time to move forward?

Thymidylate synthase is a target of 5-fluoruracil, a pyrimidine analog used to treat gastrointestinal and other cancers. The 5-fluorouracil metabolite, fluoro-deoxyuridine monophosphate, forms a ternary complex with thymidylate synthase and 5,10-methylene tetrahydrofolate. The purpose of this study was to evaluate the time-honored connection between thymidylate synthase and 5-fluorouracil. From our literature search spanning reports from 1995 to 2007 published in journals having an impact factor greater than 2, we stratified the tumors within each article, according to low versus high thymidylate synthase expression. These groups were subdivided into responders, stable disease, or disease progression. The relationship between thymidylate synthase expression and 5-fluorouracil response was analyzed for the overall group, as well as for subsets. Overall, the literature supported an approximately 2-fold inverse relationship between thymidylate synthase expression and response to 5-fluoruracil. We found no change in the trend for a relationship between thymidylate synthase and 5-fluorouracil when the literature was stratified by date of publication, impact factor of the journal in which the report was published, or substrate (mRNA versus protein) for measuring thymidylate synthase expression. Of note, there is no significant change in the trend when comparing 5-fluorouracil treatment alone or in combination with leucovorin. We found a decline of this trend when certain chemotherapeutics were used in combination with 5-fluorouracil. In sum, the connection between thymidylate synthase expression and patient response to 5-fluorouracil does not satisfy expectations for an effective drug-target relationship; and thus, studies of the thymidylate synthase tandem repeat status might only be clinically valuable in regards to patient toxicity. Thus, we question the reliability of thymidylate synthase expression as a clinical predictor of 5-fluorouracil response. Future research could perhaps be directed towards alternate targets and metabolites of 5-fluorouracil, in an effort to find a clinically relevant biomarker panel for response and to optimize fluoropyrimidine-based therapy.

Induction of monocyte chemoattractant protein-1 by nicotine in pancreatic ductal adenocarcinoma cells: Role of osteopontin

BACKGROUND Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA). We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein (MCP)-1 and evaluated the role of OPN in mediating these effects. METHODS MCP-1 mRNA and protein in PDA cells treated with or without nicotine (3-300 nmol/L) or OPN (0.15-15 nmol/L) were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Luciferase-labeled promoter studies evaluated the effects of nicotine and OPN on MCP-1 transcription. Intracellular and tissue colocalization of OPN and MCP-1 were examined by immunofluorescence and immunohistochemistry. RESULTS Nicotine treatment significantly increased MCP-1 expression in PDA cells. Interestingly, blocking OPN with siRNA or OPN antibody abolished these effects. Transient transfection of the OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P < .05) increased MCP-1 mRNA and protein and induced its promoter activity. MCP-1 was found in 60% of invasive PDA lesions, of whom 66% were smokers. MCP-1 colocalized with OPN in PDA cells and in the malignant ducts, and correlated well with higher expression levels of OPN in the tissue from patients with invasive PDA. CONCLUSION Our data suggest that cigarette smoking and nicotine may contribute to PDA inflammation by inducing MCP-1 and provide a novel insight into a unique role for OPN in mediating these effects.

Reduction of pp32 expression in poorly differentiated pancreatic ductal adenocarcinomas and intraductal papillary mucinous neoplasms with moderate dysplasia

Nuclear phosphoprotein 32 (pp32) inhibits K-ras induced transformation in experimental models. pp32 mRNA expression correlates with differentiation status in breast and prostate cancers. In this study, we evaluated pp32 protein expression in relation to the differentiation status of pancreatic ductal adenocarcinomas and precursor lesions of the pancreatic cancers. pp32 expression showed strong nuclear staining in normal pancreatic acini and ducts. The intensity of this staining was maintained in pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms with mild dysplasia, well-differentiated adenocarcinomas, and in a subset of moderately differentiated adenocarcinomas. pp32 staining was absent or reduced in poorly differentiated tumors and in intraductal papillary mucinous neoplasms with moderate dysplasia. We validated pp32 expression by a second technique, immunoblot analysis of lysates from resected pancreatic ductal adenocarcinomas and pancreatic cancer cell lines. The well-differentiated pancreatic cancer cell line HPAC expressed high amounts of pp32, as compared to the poorly differentiated pancreatic cancer cell lines MiaPaCa2, Pl19, and Pl21 cells. Artificial introduction of pp32 expression into a poorly differentiated cell line, MiaPaCa2, caused an increase in G1 arrest compared to control cells. On the basis of this study and previous functional work that shows pp32 can inhibit K-ras transformation, we propose that reduction in pp32 expression levels may be a critical event in the progression of pancreatic tumorigenesis in an aggressive subset of pancreatic ductal adenocarcinomas.