DPC4 Gene Research Articles

The Native Flip-Flop-Intrahepatic Papillary Neoplasm of Bile Duct

Intrahepatic papillary neoplasm of bile duct configures as a papillary neoplasm with a distinct fibro-vascular stalk layered by variant epithelium pervading intrahepatic bile ducts with cystic dilatation. Neoplasm configures as a premalignant lesion associated with low grade, intermediate grade or high grade intraepithelial neoplasia. Tumour progression is concordant with preliminary inactivation of TP53 and p16, antecedent activating mutations within KRAS and delayed loss of SMAD4/ DPC4 gene. Cogent clinical symptoms arising due to biliary obstruction as hyper-bilirubinaemia or abdominal pain may emerge. Cytological smears are hyper-cellular and comprised of broad, double layered sheets and aggregates of columnar epithelial cells enmeshed within a fine stroma commingled with complex, three dimensional, branching papillary configurations. Morphologically, complex tubulovillous structures or micropapillary articulations appear to distend bile ducts or enlarged, multi-locular cysts are layered by pancreaticobiliary, intestinal, gastric or oncocytic epithelium.

Post-transcriptional regulation DPC4 gene by miR-190 in colorectal cancer cells.

The objective of this study is to elucidate the regulation of the DPC4 gene by miR-190 in colorectal cancer (CRC) cells. The present study was undertaken to determine whether the DPC4 gene is a target gene of miRNA-190, identify target motifs and to elucidate the mechanism of regulation of DPC4 by miRNA-190. MiR-190 and DPC4 expression were measured in five different CRC cell lines by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The regulation of DPC4 by miR-190 was evaluated by qRT-PCR, Western blotting, and luciferase reporter assays in the human CRC cell line HT-29 after treatment with miR-190 mimics and inhibitors. The DPC4 mRNA, miR-, and DPC4 protein expression levels were highest in LS174T cells while lowest in SW480 and SW620 cells. The DPC4/miR-190 ratio in the HT-29 cancer cell line was the largest. MiR-190 expression increased dramatically after treatment with miR-190 mimics and decreased significantly after treatment with miR-190 inhibitors. DPC4 protein expression decreased in the miR-190 mimics transfection group when compared to the negative control (N.C.) group and increased in the miR-190 inhibitor groups when compared to the inhibitor plus N.C. group. MiR-190 inhibits the relative luciferase activity of psiCHECK-2™ vector-3'UTR compared to the N.C. group, while miR-190 had no obvious effect on the relative luciferase activity of the psiCHECK-2™ vector-3'UTRmut and psiCHECK-2™ vector transfected cells. The DPC4 gene might be the target gene of miR-190, which may negatively regulate the DPC4 gene in human CRC cells by translational suppression rather than mRNA degradation.

DPC4 gene expression in primary pancreatic ductal adenocarcinoma: relationship with CT characteristics.

To investigate the relationship between CT imaging findings and DPC4 gene expression and to determine the prognostic value of DPC4 gene expression to predict overall survival in patients with pancreatic ductal adenocarcinoma. Between January and December 2011, we retrospectively analyzed 163 pancreatic ductal adenocarcinomas in 163 patients who had undergone surgical resection (mean age = 61.8 years; range = 35-81 years). We divided the study patients into two groups according to DPC4 gene expression: DPC4-expression or DPC4-non-expression group. The CT findings were analyzed by two reviewers. The associations between the CT imaging findings and DPC4 gene expression were evaluated using univariate analysis and multivariate logistic regression analysis. Overall survival was compared according to the DPC4 gene expression (DPC4-expression vs DPC4-non-expression) using Kaplan-Meier analysis and log-rank testing. To avoid bias, subgroup analyses of CT findings in T3 tumour and overall survival in patients with T3 tumour and R0 resection were performed. Between DPC4-expression group (n = 75) and DPC4-non-expression group (n = 88), three CT findings (i.e., tumour margin, peripancreatic infiltration, and the presence of background intraductal pancreatic mucinous neoplasm) were significantly different in univariate analysis. Of these, a well-defined tumour margin was significantly associated with DPC4-expression tumour (adjusted odds ratio = 2.06; p = 0.032) in multivariate analysis. Of the total 163 patients, the mean overall survival of the DPC4-expression group was significantly longer than that of the DPC4-non-expression group (30.0 vs 22.0 months; p = 0.049). Of the 150 T3 tumours, the presence of well-defined tumour margins was also a significant CT finding (adjusted odd ratio = 2.00; p = 0.044) in multivariate analysis. However, of 131 patients with T3 tumour and R0 resection, the overall survival period of the DPC4-expression group was not significantly different from that of the DPC4-non-expression group (24.0 vs 22.0 months; p = 0.240). The presence of well-defined tumour margins on CT was significantly linked with DPC4-expression tumour. Advances in knowledge: A well-defined tumour margin is an independent CT finding associated with DPC4-expression pancreatic ductal adenocarcinoma.

The DPC4/SMAD4 genetic status determines recurrence patterns and treatment outcomes in resected pancreatic ductal adenocarcinoma: A prospective cohort study.

ObjectivesThe objective of this study was to investigate the role of genetic status of DPC4 in recurrence patterns of resected pancreatic ductal adenocarcinoma (PDAC).MethodsBetween April 2004 and December 2011, data on patients undergoing surgical resection for PDAC were reviewed. Genetic status of DPC4 was determined and correlated to recurrence patterns and clinical outcomes.ResultsAnalysis of 641 patients revealed that genetic status of DPC4 was associated with overall survival and was highly correlated with recurrence patterns, as inactivation of the DPC4 gene was the strongest predictor of metastatic recurrence (odds ratio = 4.28). Treatment modalities for recurrent PDAC included chemotherapy alone and concurrent chemotherapy along with local control. For both locoregional and metastatic recurrence, local control resulted in improved survival; however, for groups subdivided according to recurrence patterns and genetic status of DPC4, local control contributed to improved survival in locoregional recurrences of patients with expressed DPC4, while chemotherapy alone was sufficient for others.ConclusionsGenetic status of DPC4 contributes to the recurrence patterns following pancreatectomy, and patients with an initially expressed DPC4 gene receive a greater benefit from intensive local control for locoregional recurrence. The DPC4 gene, therefore, may aid the establishment of treatment strategies for initial adjuvant treatment or for recurrent PDAC.

Abstract IA25: Metastatic programs in pancreas cancer

Abstract The unusually high metastatic proclivity of pancreas cancer is life-limiting for a majority of patients including those treated at early stages. A minority of patients nevertheless presents with and succumbs to locally destructive disease. These distinct disease presentations and predilections for distant spread versus local growth of the primary tumor also suggest that the appropriate selection and application of systemic versus local therapies might increase their efficacy and prolong patient survival. We have undertaken a systematic effort to dissect the pathophysiologic mechanisms underlying the extreme lethality of pancreatic ductal adenocarcinoma (PDA) primarily through the generation and study of genetically engineered mouse models (GEMMs) of the disease. These models faithfully recapitulate the clinical syndrome, histopathology, molecular features and response and resistance to treatments seen in the human disease. We previously showed that point-mutant forms of Trp53 cooperate with oncogenic KrasG12D to promote widely metastatic disease in a GEMM of PDA. These and other studies have revealed that distinct combinations of tumor suppressor gene mutations can alter the pace, phenotype and prognosis of the resultant invasive disease initiated by oncogenic Kras. We have recently developed model systems that manifest the two ends of the disease phenotype described above and identified the Runx3/RUNX3 transcription factor as a master regulator of a metastatic program that dictates the balance between cell division and dissemination. Acting in concert with point-mutant Trp53 and distinct gene dosages of Dpc4/Smad4, Runx3 suppresses local growth at the expense of distant spread. Thus, Runx3 can serve as both tumor suppressor and promoter depending on the specific disease behavior in question. Runx3 promotes the invasive and migratory capabilities of PDA cells and activates a secretory program of proteins to construct a favorable microenvironment for newly migrant cells in foreign tissues such as the liver and lungs. Perhaps not surprisingly for such a potentially important behavioral switch, Runx3 levels are regulated by several inputs operating at both the transcriptional and post-translational levels. We observed a biphasic dependency of Runx3 levels on Dpc4 gene dosage in PDA which also correlates with metastatic potential. Integrating these findings, we propose a presumptive rule set to predict and explain three potential disease presentations in patients: 1) metastatic disease predominant; 2) local growth predominant; and 3) tumors with both rapid primary tumor growth and significant disseminated disease in an extraordinarily lethal combination. These three definable states also immediately suggest whether local (surgery, radiation) vs. systemic (chemotherapy) interventions should be implemented or prioritized. Thus, in addition to providing additional therapeutic targets to impede or inhibit PDA cells from establishing a clinically meaningful metastatic disease burden, this understanding has the potential to more rationally inform the specific application and timing of distinct therapeutic modalities already at hand to optimize survival. Citation Format: Sunil R. Hingorani. Metastatic programs in pancreas cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA25.

Large-scale analysis for treatment strategy according to genetic alterations of KRAS and DPC4 (SMAD4) genes in pancreatic ductal adenocarcinoma.

361 Background: To investigate correlation of genetic alterations of pancreatic ductal adenocarcinoma (PDAC) with patients’ survival, recurrence patterns, and treatment for recurrent disease. Methods: We reviewed genetic alterations of major 4 genes (K-ras, DPC4, p53, and c-erbB-2) in 699 patients who underwent surgical resection, and correlated with clinical outcomes. Results: Median survival of all patients was 21.7 months, and 5-year survival rate was 20.4%. Alterational rates of each gene were as follows: K-ras, 48.3%; DPC4, 68.1%; p53, 40.8%; c-erbB-2, 14.0%. Mutation of K-ras and inactivation of DPC4 genes were associated with shorter patients’ survival in univariate analysis. In multivariate analysis, mutation of K-ras gene was independently correlated with patients’ survival (especially, GAT and TGT subtypes). Inactivated DPC4 gene had no independent correlation with overall survival, but it was strongly associated with distant metastasis following pancreatectomy. Survival after recurrence (SAR) was evaluated in subdivided groups according to DPC4 gene function (intact or loss)/recurrence patterns (locoregional or distant)/treatment options (local control or systemic therapy). In these 8 groups, patients in intact/locoregional/local control group showed the longest survival (24.2 months of median SAR), and intact/locoregional/systemic therapy and loss/distant/systemic therapy groups were associated with much shorter survival (9.5 and 9.2 months of median SAR, respectively). Conclusions: Assessment of genetic features of PDAC may assist in deciding targeted surveillance or treatment for primary as well as recurrent PDAC, and further studies for therapeutic strategies according to genetic analyses should be devised.

MP28-08 DPC4 LOSS OF EXPRESSION IN INVASIVE UROTHELIAL CARCINOMAS: AN IMMUNOHISTOCHEMICAL STUDY

INTRODUCTION AND OBJECTIVES: DPC4 (SMAD4) is a tumor suppressor gene that encodes for a critical transcription factor involved in the TGF-beta signal pathway. Genetic inactivation of the DPC4 gene has been described in pancreatic and extrahepatic biliary adenocarcinomas, and loss of DPC4 expression by immunohistochemistry is considered a relatively specific marker of tumors originating from those sites. However, the status of DPC4 has not been previously studied in urothelial carcinomas (UC). We evaluated DPC4 expression in a set of invasive UC of the bladder and the upper urinary tract (UT). METHODS: Formalin-fixed paraffin-embedded tissues from 13 invasive UC of the upper UT (12 high grade and 1 low grade) and 18 invasive high grade UC of the bladder were retrieved from our surgical pathology archives and used to build 2 tissue microarrays. Paired tumor and non-neoplastic tissue were spotted 4 times each. DPC4 expression was evaluated using standard immunohistochemistry (DPC4: clone B8, Santa Cruz, CA). DPC4 expression in each case was categorized as intact (positive DPC4) or lost (at least focal negative DPC4). Only spots with positive internal control were considered. Intact labeling was defined as uniform expression of DPC4 in the cytoplasm of tumor cells per spot, with at least focal nuclear expression of DPC4. DPC4 loss was defined as lack of both cytoplasmic and nuclear expression in any subset of tumor cells in the presence of positive internal control staining. Several cut off values were used to reflect the extent of DPC4 loss ( 75% of the tumor cells). RESULTS: We found varying extent of DPC4 loss in 14 cases of invasive UC. Among the bladder carcinomas, 7/18 (39%) cases had some extent of DPC4 loss. Among the upper UT carcinomas, 7/13 (54%) cases had some extent of DPC4 loss, as detailed in table 1. Intact DPC4 staining was observed in 11/18 cases (61%) of invasive UC of the bladder and in 6/13 cases (46%) of invasive UC of upper UT. CONCLUSIONS: Our findings reveal that loss of DPC4 expression can be observed in UC. The current TMA based findings merrit further evaluation in whole sections of UC using immunohistochemistry. If confirmed, our findings suggest that urothelial carcinoma should be considered in the differential diagnosis of a metastatic carcinoma of unknown primary, when loss of DPC4 is seen. Sequencing analysis to assess DPC4 gene status in UC is also warranted.

Prognostic Value of DPC4 Status for Resected Pancreatic Adenocarcinoma Patients Receiving Adjuvant Chemoradiation

DPC4, a tumor suppressor gene mutated in 50% of pancreatic cancers, was strongly associated with patterns of recurrence in a previous autopsy series of advanced pancreatic cancer patients. DPC4 intact patients were found to have limited metastatic burden and more frequently died of localized disease, while DPC4 mutant patients more often harbored widely disseminated metastasis. The purpose of this study was to determine whether DPC4 gene status can predict for survival and patterns of recurrence in a cohort of resected pancreatic cancer patients treated with adjuvant chemoradiation (CRT). Patients who received adjuvant 5-FU or gemcitabine based CRT at Johns Hopkins between 1994 and 2008 with documented patterns of failure were retrospectively identified (n = 101). DPC4 status was determined from the resection specimen and graded as intact (WT) or lost/mutated (MT) by a single pathologist. Node status, margin status, disease grade, tumor size, PVI/PNI, and post-op CA19-9 level were recorded. Kaplan-Meier estimates were used to determine median overall survival (mOS) and time to progression (TTP). Cox proportional hazards models were used to compare risk factors. Median age was 61 years, and 59% of patients were male. DPC4 MT status was not associated with nodal involvement, margin positivity, or post-op CA 19-9 level (p > 0.05) when these factors were examined individually. However, DPC4 MT status was more common in patients that had all high-risk tumor features (Grade 3+, >3 cm, PVI/PNI, node +) than in those without these characteristics (82.4% vs. 47.5%, p = 0.01). DPC4 MT patients did not experience a difference in either mOS (21.9 vs. 22.6 months, p = 0.82) or TTP (13.8 vs. 14.0, p = 0.79) when compared to DPC4 WT patients. Additionally, there was no association between DPC4 MT status and mOS after adjusting for node status, margin status, tumor grade, tumor size, and age (RR 1.04, 0.62-1.74, p = 0.89). While rates of metastasis did not differ between DPC4 MT and DPC4 WT patients (55.7% vs. 62.8%, p = 0.45), local recurrence was more common in the MT subset (34.4% vs. 13.7%, p = 0.012). In resected pancreatic cancer patients receiving adjuvant CRT, DPC4 status did not predict for either mOS or TTP. However, DPC4 MT patients experienced a higher rate of local recurrence and were more likely to have high-risk disease characteristics. While these results differ from a previously reported association between DPC4 MT status and widespread metastasis, DPC4 gene status may instead be a marker for aggressiveness of disease. Additional studies that investigate the role of DPC4 across various stages and treatment regimens are necessary to define its prognostic value.

Correlation of DPC4 status with outcomes in pancreatic adenocarcinoma patients receiving adjuvant chemoradiation.

168 Background: In an autopsy series of patients with advanced pancreatic cancer (PCA), loss of DPC4 was highly correlated with disseminated metastasis. The purpose of this study was to determine if DPC4 gene status predicts for survival and patterns of recurrence following adjuvant (adj) chemoradiation (CRT). Methods: 101 patients who underwent surgery followed by adjuvant 5-FU or gemcitabine-based CRT were studied. Imaging studies were reviewed to assess patterns of recurrence and tumor tissue obtained to determine DPC4 immunolabeling status. DPC4 status was graded as intact or lost/mutated. Kaplan-Meier estimates were used to compute survival and Cox proportional hazards were used to compare risk factors. Results: Median overall (mOS) and progression-free survival (PFS) was 22.6 mos (95% CI 18.8 to 31.6) and 14.0 mos (95% CI 11.5 to 18.4). The mOS and 1-yr OS for patients with DPC4 intact vs. lost status was 21.9 mos (95% CI 16.8-32.4) and 78.4% vs. 22.6 mos (95% CI 18.4-32.6) and 78.2%, respectively (HR: 1.05, p=0.82). After adjusting for node, margin status, tumor grade, tumor size, and age, DPC4 status did not predict for mOS (RR 1.04, 95% CI: 0.62-1.74, p=0.89). Time to first progression at any site for PCA with DPC4 intact vs. lost status was 13.8 vs. 14.0 mos (p=0.79). Local recurrence was more common in PCA with DPC4 loss than with intact status (34.4% vs. 13.7%, p=0.012). There was no difference in the rates of distant recurrence in PCA with intact vs. loss of DPC4 expression (62.8% vs. 55.7%, p=0.45); however, DPC4 loss was more commonly associated with liver recurrence (27.9% vs. 19.6%, p=0.31). Conclusions: In pancreatic cancer patients receiving adj CRT, loss of DPC4 labeling in their resected PCA indicates a greater likelihood of developing local recurrence despite having received adj CRT. Efforts to improve loco-regional control are therefore needed for these patients following surgery and adj CRT. No significant financial relationships to disclose.

Effect of DPC4 gene transfection on chemotherapy sensitivity of pancreatic carcinoma cells

Objective To observe the effect of DPC4 gene transfection on the chemotherapy sensitivity of pancreatic carcinoma cells. Methods The human DPC4 complementary DNA was subcloned to the retroviral vector pLXSN to obtain recombinant pLXSN/DPC4 with direct inserting potential. The daughter cell BxPC-3/DPC4 which had DPC4 stable expression was acquired after the pancreatic carcinoma BxPC-3 cells had been transfected with pLXSN/DPC4. The sensitivity of the carcinoma cells for 5-Fu and gemcitabine was observed. Meanwhile, the mRNA level of Mdr-1 and Chk1was detected by semi-quantity PCR assay. Results The 50% inhibiting concentrations (IC50)of 5-Fuand gemcitabin4e for BxPC-3 (culturing for 72 h) were rather lower than those of BxPC-3/pLXSN and BxPC-3/-cells. Moreover, the semi-quantity PCR assay revealed that the mRNA level of Mdr-1 and Chk1 was down-regulated. These findings indicated that pLXSN/DPC4 vector, 5-Fu and gemcitabine could inhibit the growth of pancreatic cancer cells. The combined therapy with pLXSN/DPC4 vector and chemotherapeutic drugs could further inhibit the growth of cancer cells. Conclusion The DPC4 gene transfection could enhance the sensitivity of pancreatic cells to chemotherapy, which may be realized through the down-regulation of Mdr-1 and Chk1 gene expression. Key words: Pancreatic neoplasms; DPC4; Retroviral vector; Gene therapy; Chemotherapy

Abstract 1613: BTK as a drug target for cancers that harbor inactivating mutations in the DPC4 gene

Abstract The Deleted in Pancreatic Cancer locus 4 (DPC4) is a tumor suppressor gene that is often inactivated in pancreatic and colorectal cancers. In pancreatic adenocarcinomas, the frequency of inactivation of DPC4 is approximately 55%, while in colorectal carcinomas has been reported to be 10% to 35%. The loss of DPC4 has been shown to be associated with the progression and malignancy of pancreatic cancer along with decreased patient survival. We employed a small interfering (siRNA) library based screening strategy to identify potential synthetic lethal partners of the DPC4 gene. We used a kinase focused siRNA library that consisted of two siRNA oligonucleotides for each of the 624 protein kinase genes. A DPC4 isogenic pair, BxPC3-Vector and BxPC3-DPC4 (Wang et al. 2006) were treated by the siRNA oligonucleotides in parallel and the effects of the siRNA oligonucleotides on the growth of the cell lines were then compared. siRNA oligonucleotides that selectively inhibited the cell growth of the BxPC3-Vector cell line were selected as potential positive hits. These genes, once validated, represented potential drug targets that are very specific to cancer cells harboring mutations in the DPC4 gene. One of the top-ranked hits we identified from this screening is the Bruton agammaglobulinemia tyrosine kinase (BTK). With BTK siRNA treatment, BxPC3-Vector cells, which are DPC4 null, showed ∼30% more cell growth inhibition than the BxPC3-DPC4 cells, which are DPC4 wildtype. This selectivity of BTK siRNA against DPC4 null cells was further confirmed in a confirmation screen using two pancreatic cancer cell lines, BxPC3 and PANC-1, which are DPC4 null and DPC4 wildtype, respectively. BTK siRNA also showed significant selectivity against DPC4 deficiency in another pair of DPC4 isogenic colon cancer cell lines, HCT-116 and HCT-116-DPC4-knockout. To validate the BTK siRNA selectivity findings, we evaluated the BTK inhibitor PCI-32765 in our cancer cell line models and observed the same selectively against DPC4 null pancreatic and colon cancer cell lines. These results indicate that BTK is a potential molecular target for pancreas, colon, and other cancers harboring inactivating mutations in the DPC4 gene. (The work was supported by a NCI grant P01CA109552.) 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 1613.

Identification and Characterization of a Novel Anticancer Agent With Selectivity Against Deleted in Pancreatic Cancer Locus 4 (DPC4)-Deficient Pancreatic and Colon Cancer Cells

The deleted in pancreatic cancer locus 4 (DPC4)/SMAD4 tumor suppressor gene is frequently inactivated in pancreatic (approximately 55%) and colorectal cancers (approximately 30%). Like other tumor suppressor genes, the loss-of-function mutations found in the DPC4 gene are specific to cancer cells. This provides an attractive and unique opportunity for therapeutic intervention. The aim of this study was to identify and characterize small molecules that selectively kill DPC4-deficient cancer cells. An unbiased chemical screening using isogenic cell lines that only differ in the DPC4 gene was carried out to identify positive hits. Selected hits were further verified in additional isogenic cell lines and characterized in cancer cells using several different cellular assays. A lead molecule, UA62784, was identified to be selectively cytotoxic against cancer cells with deficient DPC4. UA62784 preferentially induces cell cycle arrest and apoptosis in cells with deficient DPC4. It also selectively reduces the clonogenicity of DPC4-deficient cells on soft agar when compared with cells with wild type DPC4. We further demonstrate that UA62784 induces CDC2 kinase activity preferentially in DPC4-negative cells. UA62784 is a small molecule that selectively kills DPC4-deficient cancer cells. Its potent activity and relatively low molecular weight make it a decent candidate for further lead optimization.

UA62784, a novel inhibitor of centromere protein E kinesin-like protein

Pancreatic carcinoma is the fourth leading cause of death from cancer. Novel targets and therapeutic options are needed to aid in the treatment of pancreatic cancer. The compound UA62784 is a novel fluorenone with inhibitory activity against the centromere protein E (CENP-E) kinesin-like protein. UA62784 was isolated due to its selectivity in isogenic pancreatic carcinoma cell lines with a deletion of the DPC4 gene. UA62784 causes mitotic arrest by inhibiting chromosome congression at the metaphase plate likely through inhibition of the microtubule-associated ATPase activity of CENP-E. Furthermore, CENP-E binding to kinetochores during mitosis is not affected by UA62784, suggesting that the target lies within the motor domain of CENP-E. UA62784 is a novel specific inhibitor of CENP-E and its activity suggests a potential role for antimitotic drugs in treating pancreatic carcinomas.

Inhibition of pancreatic carcinoma cell growth in vitro by DPC4 gene transfection

To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas, and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro. The expression of DPC4 was determined in 40 pancreatic adenocarcinoma and 36 non-malignant pancreatic specimens by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohisto-chemistry. Furthermore, we constructed retroviral vectors containing DPC4, which then infected the pancreatic carcinoma cell line BxPC-3. Cell growth in vitro after being infected was observed, and the vascular endothelial growth factor (VEGF) mRNA level in the daughter cells was determined by semi-quantitative PCR assay. The RT-PCR assay showed a positive rate of DPC4 mRNA in 100% (36/36) of normal specimens, compared to 40% (16/40) in adenocarcinoma specimens. The regional and intense positive cases of DPC4 expression in adenocarcinoma detected by immunohistochemistry were 10 and four, whereas it was all positive expression in normal tissues. There was a significant difference of DPC4 expression between them. The stable expression of DPC4 in the pancreatic carcinoma cells BxPC-3 could be resumed by retroviral vector pLXSN transfection, and could inhibit cell growth in vitro. Rather, DPC4 could decrease VEGF mRNA transcription levels. The deletion of DPC4 expression in pancreatic carcinoma suggests that loss of DPC4 may be involved in the development of pancreatic carcinoma. The retroviral vector pLXSN containing DPC4 can inhibit the proliferation of pancreatic carcinoma cells, and down-regulate the level of VEGF.

Effect of DPC4 Gene on Invasion and Metastasis of Colorectal Carcinoma Cells

To investigate the effect of DPC4 gene on invasion and metastasis of colorectal carcinoma cells, the expression of DPC4 was detected in sixty-three samples of colorectal tumors and seven cases of colorectal mucosa. The biological behavior of tumors expressing DPC4 was evaluated (including tumor staging, differentiation degree and metastasis). pcDNA3.1-DPC4 plasmid was constructed and transferred into HCT116 cells not expressing DPC4. The cell models (DPC4(+)-HCT116) steadily expressing DPC4 were obtained. Compared with HCT116 and pcDNA3.1-HCT116 cells, the doubling time of DPC4(+)-HCT116 cells was lengthened obviously (P<0.01), the apoptosis rate of DPC4(+)-HCT116 cells was significantly increased (PP<0.01), the cloning efficiency, cell adherency, migration and invasion ability of DPC4+-HCT116 cells were dropped obviously (P<0.01). The number of cancer nodules was decreased significantly in abdominal cavity and liver of the nude mice inoculated with DPC4(+)-HCT116 cells. The activity of MMP-9 and MMP-2 was detected by gelatin zymography. In comparison with HCT116 and pcDNA3.1-HCT116 cells, the activity of MMP-9 was decreased in DPC4(+)-HCT116 cells. Therefore, the down-regulation of DPC4 expression may be associated with the carcinogenesis of colorectal carcinoma. DPC4 may inhibit the proliferation of colon cancer cell by restraining growth and inducing apoptosis, and the invasion and metastasis of colorectal carcinoma cells. MMP-9 may be one of the downstream target genes regulated by DPC4.

In vivo platform for translational drug development and biomarker discovery in pancreatic cancer

4000 While there are many new agents entering clinical development, there is very little data to prioritize which agents should be explored in pancreas cancer. Furthermore, often there is no information on biomarkers that may predict the activity of these drugs in pancreas cancer. In this project, we have generated a cohort of 30 pancreatic cancer xenografts by implanting in nude mice tumor materials from surgical specimens. Molecular studies show that a) these tumors maintain the genetic features of the originator cancer such as KRAS, p53 and DPC4 gene status; b) tumors represent the heterogeneity of pancreatic cancer well (there is not selection of any genotype in the xenograft ) and c) features do not changed over time. We have used this platform to explore the activity of a battery with a total of 10 new anticancer agents including inhibitors of MAPK, EGFR, mTOR, src kinase, Ras oncogene, mitosis regulators, angiogenesis, heat shock protein and hedgehog pathway inhibitors using a methodology similar to a two stage phase II clinical trial. All agents are tested against 10 xenografts. Those inactive are not explored anymore. Active agents are tested against the full set of tumors. Thus far we have shown that inhibitors of mTOR and MAPK have substantial activity in this model while Ras interacting agents and EGFR targeted drugs have no single agent activity. For active agents, we have characterized the tumors for potential strategies and biormarkers that may predict activity using both a target-focus approach as well as general profiling approach. Signaling inhibitors are more active in tumors with evidence of activation of the targeted pathway. In addition, ex vivo assays indicate that the ability to inhibit the targeted pathway is associated with agent activity. This information may help to prioritize agents for clinical development in pancreatic cancer. [Table: see text]

Inherited Polyposis Syndromes: Molecular Mechanisms, Clinicopathology, and Genetic Testing

The inherited polyposis syndromes are a group of conditions in which multiple gastrointestinal polyps occur in the lumen of the gastrointestinal tract, most exhibit an increased risk of colon cancer. Benign and malignant extraintestinal tumors might also be observed. Recent elucidation of the underlying gene mutations has contributed to our understanding of the cell biology and molecular mechanisms associated with gastrointestinal tumorigenesis. Developments have also allowed genetic testing to become an integral component in accurate diagnosis, categorization, and management of inherited polyposis syndromes. In this review, we will focus on familial adenomatous polyposis, mutY human homologue-associated polyposis, Peutz-Jeghers syndrome, juvenile polyposis, and Cowden syndrome. It is essential that both physician and patient understand the benefits and limitations of genetic testing before submission of samples to the laboratory. There are many issues accompanying molecular diagnosis of cancer syndromes, and genetic counseling is an essential prelude to genetic testing.

Immunohistochemical Study of DPC4 and p53 Proteins in Gallbladder and Bile Duct Cancers

Gallbladder and bile duct carcinomas belong to the family of biliary tract tumors, but they demonstrate different clinical behavior. We evaluated a series of biliary tract carcinomas to determine whether they also had genotypic differences by analysis of the tumor suppressor genes DPC4 and p53. Twenty-one gallbladder cancers, 20 intrahepatic bile duct carcinomas, and 10 extrahepatic bile duct carcinomas were retrieved from the surgical pathology files of Kaohsiung Medical University Hospital. Sections were immunostained with monoclonal antibodies to the DPC4 and P53 proteins. Statistical differences between gallbladder cancer and bile duct carcinomas were determined using chi2 analysis or the Fisher's exact test, when appropriate. Two of the 21 gallbladder cancers (9.5%), 7 of the 20 intrahepatic bile duct carcinomas (35%), and five of the 10 extrahepatic bile duct carcinomas (50%) were negatively labeled for DPC4. The differences were significant between gallbladder carcinoma and both intrahepatic bile duct carcinomas (p = 0.023) and extrahepatic bile duct carcinomas (p = 0.012). A higher frequency of P53 overexpression was found in gallbladder cancers (61.9%) than in intrahepatic bile duct carcinomas (26.3%) (p = 0.024). This study suggests that the DPC4 gene may play a limited role in gallbladder carcinoma; however, p53 gene mutation is more frequently found in gallbladder cancers. In contrast, DPC4 deletion may be more common in bile duct carcinomas, especially in those arising from the extrahepatic bile duct. These findings support the concept that gallbladder and bile duct carcinomas are different tumors with differing etiologies and tumorigenesis.

Growth suppression of colon cancer cells in vitro by DPC4 gene expression and its mechanism

To study the effect of DPC4 gene expression on the growth of colon cancer cells and its mechanism. Expression plasmid pcDNA3.1-DPC4 was constructed and transfected into the colon cancer cell line SW620 by use of lipofectamine gene transfer technique. DPC4 protein expression was detected by Western blot and immunocytochemistry. The effect of DPC4 gene on the growth of SW620 cells was monitored by population doubling time (PDT) and cloning efficiency. The influence of DPC4 expression on p21WAF1 transcription was investigated by RT-PCR to detect p21WAF1 mRNA. Successful expression of DPC4 protein was detected in the transfected SW620 cells. Compared with the control cells, PDT (74 h) of the DPC4 expressing cells was prolonged and the cloning efficiency (21%) decreased. In addition, the mRNA level of p21(WAF1) in DPC4 transfected cells was increased. Overexpression of DPC4 gene inhibits the growth of colon cancer in vitro, and induction of p21(WAF1) expression may be an important functional aspect of DPC4.

Infrequent alteration of the DPC4 tumor suppressor gene in renal cell carcinoma

The aim of this study was to investigate the alterations in the DPC4 tumor suppressor gene in renal cell carcinoma (RCC). The study included 32 tumor specimens from Croatian patients with a diagnosis of RCC. Loss of heterozygosity (LOH) was investigated using three specific oligonucleotide primers for the three DPC4 polymorphic markers. Our investigation of mutations in the DPC4 gene was focused on exons 2, 8, 10 and 11. These exons belong to the mad homology domains 1 (exon 2) and 2 (exons 8-11). The presence of previously documented mutation in exons 2 (codon 100), 8 (codon 358), 10 (codon 412), and 11 (codon 493) was investigated by restriction fragment length polymorphism (RFLP) analysis, as a first screening method. Finally, the study was extended to search for any other type of mutation in the four selected exons by single strand conformation polymorphism (SSCP) assay. To increase heterozygosity, all 32 tumor specimens were tested with primers for three polymorphic markers. A total of 30 (94%) were heterozygous (informative). LOH at any of these markers was only revealed in four (13%) of the 30 informative samples. No tumor samples were positive for mutation in the four investigated exons analyzed by RFLP. In addition, no samples showed other types of mutation in denaturing conditions. Genetic alterations were shown only in a minority of patients, probably because mutation analysis of the DPC4 gene has only been partially covered by our work. It seems that exon 2 (belonging to the MH1 domain) and exons 8, 10, 11 (belonging to the MH2 domain) are not altered in RCC. This investigation must be extended on other exons of DPC4 for a better understanding a role of this gene in renal cell carcinoma.