Loss Of Smad4 Research Articles

Selective deletion of Smad4 in postnatal germ cells does not affect spermatogenesis or fertility in mice.

SMAD4 is the central component of canonical signaling in the transforming growth factor beta (TGFβ) superfamily. Loss of Smad4 in Sertoli cells affects the expansion of the fetal testis cords, whereas selective deletion of Smad4 in Leydig cells alone does not appreciably alter fetal or adult testis development. Loss of Smad4 in Sertoli and Leydig cells, on the other hand, leads to testicular dysgenesis, and tumor formation in mice. Within the murine testes, Smad4 is also expressed in germ cells of the seminiferous tubules. We therefore, crossed Ngn3-Cre or Stra8-Cre transgenic mice with Smad4-flox mice to generate conditional knockout animals in which Smad4 was specifically deleted in postnatal germ cells to further uncover cell type-specific requirement of Smad4. Unexpectedly, these germ-cell-knockout mice were fertile and did not exhibit any detectable abnormalities in spermatogenesis, indicating that Smad4 is not required for the production of sperm; instead, these data indicate a cell type-specific requirement of Smad4 primarily during testis development. Mol. Reprod. Dev. 83: 615-623, 2016. © 2016 Wiley Periodicals, Inc.

Reduced Expression of SMAD4 Is Associated with Poor Survival in Colon Cancer.

SMAD4 loss is associated with the development of metastases and poor prognosis. We evaluated expression of SMAD4 protein and its association with tumor characteristics, including biomarkers and outcome in terms of relapse-free survival and overall survival. We used 1,564 stage II/III colon cancer samples from PETACC-3 to evaluate SMAD4 expression by immunohistochemistry. SMAD4 protein expression was validated by assessing mRNA expression using available expression array data. SMAD4 expression was also studied on 34 adenomas and 10 colon cancer liver metastases with their primaries. Loss of SMAD4 immunoreactivity was defined as focal or diffuse. Cases without SMAD4 loss were subdivided into those with strong and weak expression. SMAD4 protein expression was informative in 1,381/1,564 cases. SMAD4 loss was found in 293/1,381 (21%) cases. Of 1,088 cases without SMAD4 loss (79%), 530 showed weak and 558 strong expression. SMAD4 loss occurred also in adenomas, but less extensively than in carcinomas. Liver metastases followed mostly the expression pattern of the primary tumor. SMAD4 loss, including weak expression, identified patients with poor survival in stage II as well as III and in both treatment arms. SMAD4 loss was less frequent in tumors with microsatellite instability and more frequent in those with loss of heterozygosity of 18q. We conclude that clonal loss of SMAD4 expression in adenomas, carcinomas, and liver metastases increases with disease progression. SMAD4 loss, and to a lesser extent weak expression, is strongly associated with poor survival regardless of stage. Clin Cancer Res; 22(12); 3037-47. ©2016 AACR.

MicroRNA-20a-5p promotes colorectal cancer invasion and metastasis by downregulating Smad4.

BackgroundTumor metastasis is one of the leading causes of poor prognosis for colorectal cancer (CRC) patients. Loss of Smad4 contributes to aggression process in many human cancers. However, the underlying precise mechanism of aberrant Smad4 expression in CRC development is still little known.ResultsmiR-20a-5p negatively regulated Smad4 by directly targeting its 3′UTR in human colorectal cancer cells. miR-20a-5p not only promoted CRC cells aggression capacity in vitro and liver metastasis in vivo, but also promoted the epithelial-to-mesenchymal transition process by downregulating Smad4 expression. In addition, tissue microarray analysis obtained from 544 CRC patients’ clinical characters showed that miR-20a-5p was upregulated in human CRC tissues, especially in the tissues with metastasis. High level of miR-20a-5p predicted poor prognosis in CRC patients.MethodsFive miRNA target prediction programs were applied to identify potential miRNA(s) that target(s) Smad4 in CRC. Luciferase reporter assay and transfection technique were used to validate the correlation between miR-20a-5p and Smad4 in CRC. Wound healing, transwell and tumorigenesis assays were used to explore the function of miR-20a-5p and Smad4 in CRC progression in vitro and in vivo. The association between miR-20a-5p expression and the prognosis of CRC patients was evaluated by Kaplan–Meier analysis and multivariate cox proportional hazard analyses based on tissue microarray data.ConclusionsmiR-20a-5p, as an onco-miRNA, promoted the invasion and metastasis ability by suppressing Smad4 expression in CRC cells, and high miR-20a-5p predicted poor prognosis for CRC patients, providing a novel and promising therapeutic target in human colorectal cancer.

Loss of Chromosome 18 in Neuroendocrine Tumors of the Small Intestine: The Enigma Remains

Background/Aims: Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. Methods: We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/- and Chr18+/+ SI-NETs. Results: Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/- and Chr18+/+ SI-NETs. Conclusion: DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18.

Gastric Lgr5(+) stem cells are the cellular origin of invasive intestinal-type gastric cancer in mice.

The cellular origin of gastric cancer remains elusive. Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is the first identified marker of gastric stem cells. However, the role of Lgr5(+) stem cells in driving malignant gastric cancer is not fully validated. Here, we deleted Smad4 and PTEN in murine gastric Lgr5(+) stem cells by the inducible Cre-LoxP system and marked mutant Lgr5(+) stem cells and their progeny with Cre-reporter Rosa26(tdTomato). Rapid onset and progression from microadenoma and macroscopic adenoma to invasive intestinal-type gastric cancer (IGC) were found in the gastric antrum with the loss of Smad4 and PTEN. In addition, invasive IGC developed at the murine gastro-forestomach junction, where a few Lgr5(+) stem cells reside. In contrast, Smad4 and PTEN deletions in differentiated cells, including antral parietal cells, pit cells and corpus Lgr5(+) chief cells, failed to initiate tumor growth. Furthermore, mutant Lgr5(+) cells were involved in IGC growth and progression. In the TCGA (The Cancer Genome Atlas) database, an increase in LGR5 expression was manifested in the human IGC that occurred at the gastric antrum and gastro-esophageal junction. In addition, the concurrent deletion of SMAD4 and PTEN, as well as their reduced expression and deregulated downstream pathways, were associated with human IGC. Thus, we demonstrated that gastric Lgr5(+) stem cells were cancer-initiating cells and might act as cancer-propagating cells to contribute to malignant progression.

Abstract B32: Transgenic model for early prostate cancer metastasis to the lymph nodes

Abstract The emergence of castration-recurrent (CR), metastatic prostate cancer (PC) following the failure of androgen-deprivation therapy represents the lethal phenotype of this disease. However, the genes and pathways that drive metastasis, and whether metastasis is an early or late event, remain poorly understood. Early dissemination of PC to the bones (10) and lymph nodes (8,13) has been described, and at least one study shows that a lower-grade focus, rather than high-grade foci in the same primary PC, was the source for metastases in one patient (7), suggesting collectively that metastatic dissemination is likely early event. Thus, we sought to develop a transgenic model to study early metastatic dissemination of PC. Although PTEN loss, associated with activation of AKT signaling, occurs frequently in PC, there is no increased association with metastatic disease (6,12,14), strongly suggesting that it is not sufficient as a driver of metastasis. Moreover, prostate-specific Pten loss in most mouse transgenic (Tg) models induces prostatic intraepithelial neoplasia (PIN), whereas its combination with a second relevant event, such as Myc amplification (5) or Smad4 loss (3), results in the production of aggressive primary and metastatic lesions. To address what might be the genetic drivers of early PC metastasis, we produced a transgenic mouse model containing losses of the tumor suppressor, Rb1, and the metastasis-suppressor, SSeCKS/Akap12. The loss of Rb1 signaling, resulting from either mutation or deletion of Rb1, or from the downregulation of Rb1 mediators, occurs in at least 40% of PC metastases, whereas its loss in primary PC is under 10% (6,11,12). Additionally, the downregulation AKAP12 is greater in PC metastases compared to primary PC (4,14), and the loss of Akap12 in Tg mice causes AKT activation (1) as well as Rb-dependent premature senescence (2). Whereas the loss of Rb1 or Akap12 alone resulted in prostatic hyperplasia (1,9), their combined loss resulted in high-grade (HG) PIN that fails to progress to malignancy after 18 months. Most significantly, mice with HG-PIN lesions exhibited metastases to draining lymph nodes, marked by relatively differentiated tumor cells co-expressing markers of basal (p63, cytokeratin 14) and luminal (cytokeratin 8 and androgen receptor) epithelial cells, and although none expressed the basal marker, cytokeratin 5, about 40% expressed vimentin, a marker of epithelial-to-mesenchymal transition. These cells likely arose from small, rare foci of p63-positive basal cells in the HG-PIN lesions showing similar sets of transitional markers as well as increased co-staining with nuclear androgen receptor. Interestingly, although all the PIN lesion and metastatic cells expressed upregulated levels of activated AKT (increased AKTpoS473 relative to total AKT) compared to prostate epithelial cells from Rb- or Akap12-deficient mice, the double Rb/Akap12 knockout cells did not progress to malignancy, as do Pten/Rb-deficient prostate cells, suggesting that Pten loss affects drivers in addition to AKT. Lastly, the double Rb/Akap12-deficient metastatic cells exhibited increased levels of Smad4 protein compared to single Rb- or Akap12-deficient cells, suggesting that its continued expression suppressed malignant progression. Taken together, these data suggest that in the context of Rb loss, Akap12 suppresses the oncogenic proliferation and early metastatic spread of basal-luminal prostate tumor cells. Experiments will be described to use this model to dissect the genomic drivers of early prostate cancer metastatic dissemination, and following the loss of Smad4, to identify drivers of prostate cancer malignant progression. 1. S. Akakura et al., Cancer Res. 68(13), 5096 (2008). 2. S. Akakura et al., Cell Cycle. 9(23), 4656 (2010). 3. Z. Ding et al., Nature. 470(7333), 269 (2011). 4. I.H. Gelman Cancer Metastasis Rev. 31(3-4), 493 (2012). 5. M.M. Grabowska et al., Cancer Met. Rev. 33, 377 (2014). 6. C.S. Grasso, et al., Nature. 487(7406), 239 (2012). 7. M. C. Haffner, et al., J Clin. Invest. 123(11), 4918 (2013). 8. C. A. Klein, et al., Lancet. 360(9334), 683 (2002). 9. L. A. Maddison, et al., Cancer Res. 64(17), 6018 (2004). 10. S. W. Melchior, et al., Clin Cancer Res 3(2), 249 (1997). 11. M. L. Nickerson, et al., Hum. Mutat. 34(9), 1231 (2013). 12. D. Robinson, et al., Cell. 161(5), 1215 (2015). 13. D. Schilling, et al., Prostate. 70(10), 1110 (2010). 14. B. S. Taylor, et al., Cancer Cell. 18(1), 11 (2010). Citation Format: Hyun-Kyung Ko, Shin AKakura, Jennifer Peresie, David Goodrich, Barbara Foster, Irwin H. Gelman. Transgenic model for early prostate cancer metastasis to the lymph nodes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B32.

Abstract P3-05-06: Evolution of genomic alterations on endocrine therapy and mTOR inhibition in estrogen receptor (ER)-positive breast cancer

Abstract Background: Endocrine therapy (ET) and mTOR inhibition are important treatment strategies in ER-positive breast cancer, but no specific genomic alterations reliably predict benefit. Because tumors are tested pretreatment, we hypothesized that this may not capture tumor interaction with therapy. Methods: We studied tumors from protocol NCT00570921 using fulvestrant and everolimus for metastatic ER-positive breast cancer after aromatase inhibitor (AI) failure. DNA from FFPE tumor tissue was subjected to next-generation genomic profiling using the FoundationOne® assay and alterations were then compared between available paired samples: primary/metastatic, before/after everolimus, and upon progression on everolimus. Results: The most common alterations encountered were in the PI3K/AKT/mTOR pathway with increased frequency of PIK3CA in endocrine-sensitive disease but no specific association with everolimus benefit (Massarweh et al, ASCO 2015). One patient with lobular carcinoma relapse on ET and a new contralateral primary, had PIK3CA, CDH1, and MAP3K1 in both tumors with no new alterations detected. Her disease was everolimus sensitive. Another patient with PIK3CA at baseline acquired a CTNNA1 mutation upon relapse with no everolimus benefit. Interestingly, one patient with liver metastasis and complete response to everolimus lasting 3 years had no known alterations reported in the primary tumor but had a PIK3CA mutation in one of two simultaneous biopsies of separate liver lesions. Another patient with liver metastasis and a GATA3 mutation at baseline had response to everolimus lasting 18 months, then developed a PIK3R2_c.1936A>T mutation on progression reported as a variant of unknown significance (VUS). Another patient with metastatic lobular carcinoma to skin and bone had PIK3CA, CDH1, and ERBB2 mutations at baseline, and acquired KRAS and MCL1 amplification on two sequential skin biopsies in the first month on everolimus. She remained on therapy for 1 year. One patient with locally advanced disease and de novo bone metastasis had TP53 and GATA3 mutations at baseline with resistance to multiple chemotherapy and endocrine treatments. Upon progression on AI, her tumor acquired PDGFRA and SMAD4, detected on day 1 biopsy of everolimus treatment. Repeat biopsy on day 28 revealed loss of PDGFRA and SMAD4 with emergence of PIK3CA and MLL2 mutations and loss of STK11. After 1 year on everolimus her tumor progressed and repeat breast biopsy revealed loss of the PIK3CA, STK11, and MLL2 events, with appearance of AKT1 and NF1 mutations. Interestingly, her tumor also acquired ESR1_c.1607T>G, MTOR_c.6104C>T, and NSD1_c.5938G>A mutations, all classified as VUS but were not previously encountered in her course. Further analysis and biologic relevance of these changes will be presented. Conclusion: This small study suggests that ER-positive breast cancer is a dynamic disease with genomic evolution on endocrine therapy and mTOR inhibition. In some patients, this change occurs early on therapy, possibly through clonal selection, but may also be related to tumor heterogeneity. The significance of this change is not fully understood, but study of early on-treatment biopsies may help us better understand tumor response to therapy. Citation Format: Massarweh S, Romond E, Stewart R, Sun J, Chmielecki J, Mehdi M, Black EP. Evolution of genomic alterations on endocrine therapy and mTOR inhibition in estrogen receptor (ER)-positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-06.

Integrated Genomic Analysis of Pancreatic Ductal Adenocarcinomas Reveals Genomic Rearrangement Events as Significant Drivers of Disease.

Many somatic mutations have been detected in pancreatic ductal adenocarcinoma (PDAC), leading to the identification of some key drivers of disease progression, but the involvement of large genomic rearrangements has often been overlooked. In this study, we performed mate pair sequencing (MPseq) on genomic DNA from 24 PDAC tumors, including 15 laser-captured microdissected PDAC and 9 patient-derived xenografts, to identify genome-wide rearrangements. Large genomic rearrangements with intragenic breakpoints altering key regulatory genes involved in PDAC progression were detected in all tumors. SMAD4, ZNF521, and FHIT were among the most frequently hit genes. Conversely, commonly reported genes with copy number gains, including MYC and GATA6, were frequently observed in the absence of direct intragenic breakpoints, suggesting a requirement for sustaining oncogenic function during PDAC progression. Integration of data from MPseq, exome sequencing, and transcriptome analysis of primary PDAC cases identified limited overlap in genes affected by both rearrangements and point mutations. However, significant overlap was observed in major PDAC-associated signaling pathways, with all PDAC exhibiting reduced SMAD4 expression, reduced SMAD-dependent TGFβ signaling, and increased WNT and Hedgehog signaling. The frequent loss of SMAD4 and FHIT due to genomic rearrangements strongly implicates these genes as key drivers of PDAC, thus highlighting the strengths of an integrated genomic and transcriptomic approach for identifying mechanisms underlying disease initiation and progression.

Prognostic Value of SMAD4 in Pancreatic Cancer: A Meta-Analysis

PURPOSE: The prognostic value of SMAD4 in pancreatic cancer has been evaluated in several studies. However, the conclusions remain controversial. Therefore, we aimed to evaluate the association between SMAD4 expression and the outcome of pancreatic cancer patients by performing a meta-analysis. METHODS: We systematically searched for relevant studies evaluating the relationship between SMAD4 expression and the outcome of pancreatic cancer patients until May 2015. A meta-analysis was performed using STATA 12.0, and pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate the strength of the association between SMAD4 expression and the prognosis of pancreatic cancer patients. RESULTS: The analysis included 1762 patients from 14 studies, with 1401 patients from 11 studies and 927 patients from 8 studies included in the univariate and multivariate analyses, respectively. Loss of SMAD4 expression was found to be significantly correlated with poor overall survival, with the combined HR (95% CI) of 1.20 (1.03-1.40). After adjusting for potential confounders using the Cox regression model, the pooled HR (95% CI) was 1.88 (1.31-2.70). In subgroup analysis, study region, number of patients, follow-up duration, and cutoff value were found to affect the significance of the association between loss of SMAD4 expression and poor prognosis. In addition, there was no evidence of publication bias, as suggested by Begg’s and Egger’s test. CONCLUSIONS: Loss of SMAD4 was associated with poor survival and was a negative prognostic indicator in patients with pancreatic cancer.

Loss of SMAD4 Promotes Colorectal Cancer Progression by Accumulation of Myeloid-Derived Suppressor Cells through the CCL15–CCR1 Chemokine Axis

We previously reported that loss of SMAD4 promotes chemokine CCL15 expression to recruit CCR1(+) myeloid cells via the CCL15-CCR1 axis, which facilitates metastasis of colorectal cancer to the liver. The purposes of this study were to investigate whether essentially the same mechanism works in tumor invasion of the primary colorectal cancer and to evaluate the clinical importance of CCL15 expression and CCR1(+) cell accumulation. Using human colorectal cancer cell lines with reduced expression of SMAD4 or CCL15, we investigated tumor growth activities in vivo. We used immunohistochemistry (IHC) to investigate expression of SMAD4, CCL15, and CCR1 with 333 clinical specimens of primary colorectal cancer. We next characterized the CCR1(+) cells using double immunofluorescence staining with several specific cell-type markers. Finally, we determined the serum CCL15 levels in 132 colorectal cancer patients. In an orthotopic xenograft model, CCL15 secreted from SMAD4-deficient colorectal cancer cells recruited CCR1(+) cells, resulting in aggressive tumor growth. IHC indicated that loss of SMAD4 was significantly associated with CCL15 expression, and that CCL15-positive primary colorectal cancers recruited approximately 2.2 times more numbers of CCR1(+) cells at their invasion front than CCL15-negative colorectal cancers. Importantly, these CCR1(+) cells were of the myeloid-derived suppressor cell (MDSC) phenotype (CD11b(+), CD33(+), and HLA-DR(-)). Most CCR1(+) cells showed the granulocytic-MDSC phenotype (CD15(+)), whereas some showed the monocytic-MDSC phenotype (CD14(+)). Serum CCL15 levels in colorectal cancer patients were significantly higher than in controls. Blocking the recruitment of CCR1(+) MDSCs may represent a novel molecular-targeted therapy, and serum CCL15 concentration can be a novel biomarker for colorectal cancer.

Carcinoma Associated Fibroblast: a Paradoxical Role in Pancreatic Cancer Microenvironment and a Promising Target for Therapy

Pancreatic cancer is a grave malignancy showing an upward trend in morbidity and mortality during the recent decades. For reasons of late diagnosis, chemoresistance, low potential respectable rate and high post-operative recurrence rate, it has been the 6th most common cause of cancer death in China. As one of the most aggressive malignancies and the most common type of pancreatic cancer, pancreatic adenocarcinoma (PDAC) represents a significant therapeutic challenge. Conventional chemotherapeutic cytotoxic agents are proved to be with a poor survival benefit. Though the current first-line therapy FOLFIRINOX increased the median survival time compared with gemcitabine, it has been still unsatisfactory. Another direction enlighted by the treatement experience in other tumors is targeting certain molecules that participate in specific signaling pathways mediating cancer cell proliferating, angiogenesis, chemoresistance or metastasis. Unfortunately, none of the established targeted therapy agents that have been approved to be effective in some other tumors has a similar effect on PDAC, suggesting that there are some unique and decisive elements in the microenvironment of PDAC to facilitate its extensive drugresistance. Thus, the spotlight has been turned on immunotherapy, which is theoretically curative regardless of the complex molecular and cellular heterogeneity while the concrete strategies on PDAC are still in the dark. Revisiting the complex biology of PDAC, three prime characteristics will never be missed: almost 90% of patients with oncogene mutation of KRAS, as well as loss of tumor suppressor genesTP53 and SMAD4; mostly hypovascular; and tumor desmoplasia by persistent activation of fibroblasts/ pancreatic stellate cells (PSC). The last one, which is the defining feature of PDAC, as the target of therapy is the focus of this review.

Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression.

The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival.

Smad4 sensitizes colorectal cancer to 5-fluorouracil through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade.

5-Fluorouracil (5-FU), a cell cycle-specific antimetabolite, is one of the most commonly used chemotherapeutic agents for colorectal cancer (CRC). Yet, resistance to 5-FU-based chemotherapy is still an obstacle to the treatment of this malignancy. Mutation or loss of Smad4 in CRC is pivotal for chemoresistance. However, the mechanism by which Smad4 regulates the chemosensitivity of CRC remains unclear. In the present study, we investigated the role of Smad4 in the chemosensitivity of CRC to 5-FU, and whether Smad4-regulated cell cycle arrest is involved in 5-FU chemoresistance. We used Smad4-expressing CT26 and Smad4-null SW620 cell lines as experimental models, by knockdown or transgenic overexpression. Cells or tumors were treated with 5-FU to determine chemosensitivity by cell growth, tumorigenicity assay and a mouse model. Cell cycle distribution was examined with flow cytometric analysis, and cell cycle-related proteins were examined by western blotting. Smad4 deficiency in CT26 and SW620 cells induced chemoresistance to 5-FU both in vitro and in vivo. Smad4 deficiency attenuated G1 or G2 cell cycle arrest by activating the PI3K/Akt/CDC2/survivin pathway. The PI3K inhibitor, LY294002, reversed the activation of the Akt/CDC2/survivin cascade in the Smad4-deficient cells, while it had little effect on cells with high Smad4 expression. In conclusion, we discovered a novel mechanism mediated by Smad4 to trigger 5-FU chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade. The present study also implies that LY294002 has potential therapeutic value to reverse the chemosensitivity of CRC with low Smad4 expression.

Insights Into SMAD4 Loss in Pancreatic Cancer From Inducible Restoration of TGF-β Signaling.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer death in the United States. The TGF-β signaling protein SMAD family member 4 is lost in 60% of PDAC, and this has been associated with poorer prognosis. However, the mechanisms by which SMAD4 loss promotes PDAC development are not fully understood. We expressed SMAD4 in human PDAC cell lines BxPC3 and CFPAC1 by selection of stable clones containing an inducible SMAD4 tetracycline inducible expression system construct. After 24 hours of SMAD4 expression, TGF-β signaling-dependent G1 arrest was observed in BxPC3 cells with an increase in the G1 phase fraction from 48.9% to 71.5%. Inhibition of cyclin-dependent kinase inhibitor 1A by small interfering RNA eliminated the antiproliferative effect, indicating that up-regulation of cyclin-dependent kinase inhibitor 1A/p21 by TGF-β signaling is necessary for the phenotype. SMAD4 expression had no impact on invasion in BxPC3 cells, but reduced migration. Microarray analysis of gene expression at 8, 24, and 48 hours after SMAD4 expression characterized the regulatory impact of SMAD4 expression in a SMAD4-null PDAC cell line and identified novel targets of TGF-β signaling. Among the novel TGF-β targets identified are anthrax toxin receptor 2 (3.58× at 8 h), tubulin, β-3 class III (7.35× at 8 h), cell migration inducing protein, hyaluronan binding (8.07× at 8 h), IL-1 receptor-like 1 (0.403× at 8 h), regulator of G protein signaling 4 (0.293× at 8 h), and THAP domain containing 11 (0.262× at 8 h). The gene expression changes we observed upon restoration of TGF-β signaling provide numerous new targets for future investigations into PDAC biology and progression.

Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts

The value of next-generation sequencing (NGS) of pancreatic cyst fluid relative to the clinical and imaging impression has not been well-studied. The aim of this study was to assess the impact of NGS on the clinical diagnosis from imaging and carcinoembryonic antigen (CEA) and thus the management of pancreatic cysts. Ninety-two pancreatic cyst fluids from 86 patients were analyzed by cytology, CEA, and targeted NGS. Cysts were classified by imaging as nonmucinous, mucinous, or not specified. NGS results were compared with the imaging impression stratified by CEA and cytology. NGS impacted the clinical diagnosis by defining a cyst as mucinous in 48% of cysts without elevated CEA levels. The VHL gene in 2 intraductal papillary mucinous neoplasms (IPMNs) supported a serous cystadenoma. Twenty percent of cysts that were nonmucinous by imaging were mucinous by NGS. Of the 14 not-specific cysts, CEA levels were not elevated in 12 (86%), and NGS established a mucinous etiology in 3 (25%). A KRAS or GNAS mutation supported an IPMN with nonmucinous CEA in 71%. A KRAS mutation reclassified 19% of nonneoplastic cysts with nonmucinous CEA as mucinous. Seven cyst fluids (8%) had either a TP53 mutation or loss of CDKN2A or SMAD4 in addition to KRAS and/or GNAS mutations; 5 of 7 (71%) were clinically malignant, and high-grade cytology was detected in all 5. Overall, CEA was more specific for a mucinous etiology (100%), but NGS was more sensitive (86% vs 57%). NGS of pancreatic cyst fluid impacts clinical diagnosis and patient management by defining, supporting, or changing the clinical diagnosis based on imaging and CEA. NGS was most valuable in identifying mucinous cysts with nonmucinous CEA. An added benefit is the potential to detect mutations late in the progression to malignancy that may increase the risk classification of the cyst based on imaging and cytology.

Abstract 2361: TGFβ signaling deficiency enhances tumor associated inflammation in colon cancer

Abstract In early colon cancer development, there is considerable evidence suggesting that transforming growth factor-β (TGFβ) serves as a tumor suppressor due to its growth-inhibitory effects in the epithelium. This is in contrast to observations suggesting that in advanced disease, TGFβ promotes tumor progression by contributing to metastasis. Therefore, to better understand the effects of TGFβ signaling on colon cancer, we examined the effect of global or epithelial reduction in TGFβ signaling on tumor progression in the APC murine model of colon cancer. Initially, we examined the relationship between TGFβ signaling and inflammation in human colon cancer specimens. We found that loss of SMAD4 in epithelial cells correlates with increased inflammation in tissue from colon cancer patients. Moreover, APC mice with global TGFBR deficiency (ATG mice) demonstrated increased inflammation that was also associated with loss of SMAD4 in epithelial cells. In contrast, APC mice with epithelial loss of TGFBR signaling (ATC mice) do not demonstrate loss of SMAD4, but have even more pronounced inflammation concomitant with rectal bleeding and prolapse, significant weight loss, the presence of crypt abscesses and lymphoid aggregates, and increased mortality. Similarly, ATC mice, in contrast to the ATG and the APC mice, show markedly increased tumor infiltrating macrophages, splenic macrophages, and serum levels of the cancer-associated cytokines IL8, TGFβ1, and TNFα. Overall, these findings demonstrate that loss of TGFβ signaling, particularly in epithelial cells, dramatically worsens tumor progression. Yet, the attempt to reduce metastasis by treating colon cancer patients with TGFβ inhibitors may result in worse outcome by invoking a robust inflammatory response. Citation Format: Daniel R. Principe, Brian DeCant, Riley J. Mangan, Elizabeth A. Wayne, Andrew M. Diaz, Dominic Vitello, Hidayatullah G. Munshi, Paul Grippo, Barbara Jung. TGFβ signaling deficiency enhances tumor associated inflammation in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2361. doi:10.1158/1538-7445.AM2015-2361

Abstract 2716: Epstein-Barr virus-encoded BARF1 downregulates SMAD4 and increases miR-146a in gastric carcinoma cells

Abstract Recently, molecular analytic data from ‘The Cancer Genome Atlas’ (TCGA) (http://www.cbioportal.org) has re-confirmed that Epstein-Barr virus (EBV)-infected gastric carcinoma is a specialized subset of gastric carcinomas. However, viral oncogene has not yet been established in gastric carcinomas. Several reports have suggested that EBV-encoded BARF1 may be a putative viral oncogene in gastric carcinoma. The aim of this study was to investigate alterations of cellular microRNA and cellular protein by BARF1 in gastric carcinoma cells. We cloned BARF1 from naturally EBV-infected gastric carcinoma cell line, and transfected BARF1 into EBV-negative gastric carcinoma cells, thus produced a stable cell line of BARF1-transfected gastric carcinoma cells. BARF1-transfected gastric carcinoma cells revealed significantly increased cellular proliferation than its original cells. The MicroRNA array (Agilent release 16.0) data and Taqman RT-PCR results showed that cellular miR-146a was highly expressed in BARF1-transfected gastric carcinoma cells, comparing to that in original cells. In BARF1-transfected gastric carcinoma cells, transfection with BARF1-siRNA offset the increment effect of miR-146. Using bioinformatics analyses for miR-146a target (RNA hybrid 2.2 and TargetScan 5.2 sites), we found that miR-146a target seed sequence existed in the 3′ UTR of SMAD4 mRNA. The Western blot assay demonstrated that SMAD4 was remarkably reduced by BARF1 transfection. When immunohistochemistry for SMAD4 was performed on 343 cases of surgically resected, human gastric carcinoma tissues, 32 (9%) EBV-infected gastric carcinoma group disclosed more frequent loss of SMAD4 than EBV-negative group. In conclusion, EBV-encoded BARF1 could promote proliferation of gastric carcinoma cells, thereby contributing to EBV-induced cancer progression, in which SMAD4 downregulation, probably through miR-146a increase, may be entailed. Citation Format: Dong Ha Kim, Chan Jin Yoon, Jin, Kyung Rho, Jaap M. Middeldorp, Jun Hee Woo, Mee Soo Chang. Epstein-Barr virus-encoded BARF1 downregulates SMAD4 and increases miR-146a in gastric carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2716. doi:10.1158/1538-7445.AM2015-2716

Abstract A17: The role of post-translational activation of MYC in pancreatic cancer development and progression

Abstract Pancreatic cancer is the 4th leading cause of cancer-related deaths in the United States, with 46,420 new cases and 39,590 deaths estimated in 2014. Pancreatic cancer is a disease with a high rate of mortality, as it is generally diagnosed in the advanced stages when no curative treatments are available. Despite increased understanding of the morphology, biology, and molecular aberrations in pancreatic cancer, the 5-year survival rate has not improved significantly in the past 30 years. The c-MYC (MYC) oncoprotein is a potent regulator of cell fate decisions that drives nearly all aspects of tumorigenesis, including tumor cell proliferation and tumor microenvironment remodeling. High levels of MYC expression occur in a wide variety of human tumors, and animal models demonstrate that MYC overexpression can drive tumorigenesis in many tissues. The MYC gene is frequently amplified in human pancreatic cancer and MYC mRNA and/or protein are often overexpressed. However, the role of MYC in pancreatic cancer development and progression is not well understood. Our laboratory has demonstrated that MYC is post-translationally activated by KRAS signaling. Specifically, the RAS-RAF-MEK-ERK signaling pathway leads to phosphorylation of MYC at Serine 62 (pS62-MYC), which increases MYC’s protein stability as well as its activation of oncogenic target gene expression. Furthermore, we have observed high expression of pS62-MYC in primary pancreatic tumors, starting as early as PanIN 1, compared to normal adjacent tissue. Since activating mutations in KRAS are an early and nearly universal event in the development of pancreatic ductal adenocarcinoma (PDAC), these data suggest that MYC may be a critical downstream effector of KRAS-driven pancreatic cancer. We have generated unique c-MYC knock-in mice in which MYC is expressed from the ROSA26 locus following tissue-specific expression of Cre recombinase (ROSA-LSL-MYC). These mice express transcriptionally deregulated, but physiological levels of MYC. At these levels, however, MYCWT knock-in is not sufficient to drive tumors in any tested tissue. Since KRAS can post-translationally activate MYC, we hypothesized that low-level expression of deregulated MYC would increase KRAS driven pancreatic tumors in mice that co-express KRASG12D and MYCWT . We crossed ROSA-LSL-MYC mice with LSL-KRASG12D;Pdx1-Cre (KC) mice to generate the LSL-KRASG12D;ROSA-LSL-MYC;Pdx1-Cre (KMC) model. We found that co-expression of MYC and activated KRAS induces acinar to ductal metaplasia (ADM) and accelerates both PanIN precursor lesion formation and its conversion to PDAC relative to KRAS only. Tumors from KMC mice histologically resemble human PDAC, with a 20% metastatic rate to the liver. Together, these results suggest that MYC is important for both the initiation and progression of pancreatic tumors. In addition to ductal adenocarcinoma, the MYC/KRAS expressing mice also developed metastatic, poorly differentiated carcinomas with neuroendocrine features. While activation of KRAS is thought to be one of the earliest oncogenic changes in PDAC-precursor cells, studies suggest that several other genetic events occur during the evolution of PanIN lesions to PDAC, including loss of p16, mutation/loss of p53, and loss of SMAD4. Importantly, tumors arising in our KMC model spontaneously exhibit these stage-appropriate molecular aberrations, suggesting that this model accurately recapitulates the genetic evolution of human pancreatic cancers. We also observe a strong desmoplastic reaction with prominent stroma and immune cell infiltration in these spontaneous pancreatic tumors. Thus, our novel KMC mice may provide a physiologically relevant model of human pancreatic cancer for the testing of new therapeutic agents. Citation Format: Amy S. Farrell, Colin J. Daniel, Zina Jenny, Brett Sheppard, Jody Hooper, Rosalie C. Sears. The role of post-translational activation of MYC in pancreatic cancer development and progression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A17.

Abstract A21: Stromal-derived TGFβ facilitates pancreatic cancer development via repression of T-cell mediated cytotoxicity

Abstract The roles of TGFβ in tumor development are seemingly paradoxical. Despite evidence suggesting TGFβ functions as an early stage tumor suppressor, TGFβ promotes the progression of many advanced cancers. For this reason, the effects of TGFβ across non-epithelial cell compartments were evaluated in the greater context of pancreatic cancer. Murine models expressing the human mutant KRASG12D allele (EL-KRAS) were crossed to either mice with epithelial expression of a truncated TGFBR2 (MT-Tgfbr2-DNR), or those with a global deficiency of TGFBR1 (Tgfbr1+/-). Epithelial TGFβ signaling was examined via western blotting and immunoprecipitation of protein lysates from human and mouse cell lines. Co-cultures of human pancreatic epithelial and stellate cells were established to assess paracrine TGFβ signaling with western blotting and ELISA analyses. Additionally, mouse tissue was collected and stained via immunohistochemistry or immunofluorescence. To examine the effects of TGFβ on the lymphoid compartment, mouse mesenteric lymph node was collected and subjected to flow cytometry. Pancreas specific immune activity was subsequently confirmed by immunostaining. In addition, 80 human pancreatic cancer patient samples were subject to immunohistochemical staining of SMAD4, p21, and GranzymeB and analyzed by two-way ANOVA. Despite similar disruption of epithelial TGFβ signaling in both models, the EL-KRAS/Tgfbr1+/- cohort exhibited protection against Kras-induced neoplasia, contrasting advanced disease in EL-KRAS/MT-Tgfbr2-DNR mice. These results suggest TGFBR-deficiency in a non-epithelial cell compartment attenuates the loss of tumor suppressive signaling in the epithelium. Co-cultures of pancreatic epithelial and stellate cells were established, indicating stromal overexpression of TGFβ1 in the presence of cancer epithelia. This stromal-derived TGFβ reduced lesion-targeted, cytotoxic CD8+ T-cell responses mediated by GranzymeB activation of caspase-3. The interruption of this mechanism in EL-KRAS/Tgfbr1+/- restores functional cytotoxicity, overpowering the tumor permissive phenotype of TGFBR1 reduction in pancreatic epithelium. In preparation for translation to the clinic, pancreatic cancer patients were examined for biomarkers of epithelial and tumor microenvironment TGFβ signaling. Statistical analysis revealed no significant correlation between the tumor suppressive SMAD/p21 axis and GranzymeB deposition, indicating cytotoxic responses may be restored following TGFβ inhibition in patients with SMAD4 loss in the epithelia. In summary, global deficiency of TGFBR1 restores a cytotoxic T-lymphocyte response by interrupting stromal overexpression of TGFβ, thereby protecting against mutant Kras-induced neoplasia. Given this protection, patients with simultaneous SMAD4 and GranzymeB deficiencies may be optimal candidates for inhibition strategies targeting the TGFβ pathway. Citation Format: Brian DeCant, Daniel Principe, Elizabeth Wayne, Emman Mascarinas, Hidayatullah Munshi, Barbara Jung, Paul Grippo. Stromal-derived TGFβ facilitates pancreatic cancer development via repression of T-cell mediated cytotoxicity. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A21.

Smad1/5 and Smad4 expression are important for osteoclast differentiation.

To investigate the necessity of the canonical BMP pathway during osteoclast differentiation, we created osteoclasts with a conditional gene deletion for Smad1 and Smad5 (SMAD1/5), or Smad4 using adenovirus expressing CRE recombinase (Ad-CRE). Reduction of either Smad4 or Smad1/5 expression resulted in fewer and smaller multinuclear cells compared to control cells. We also detected changes in osteoclast enriched genes, demonstrated by decreased Dc-stamp and cathepsin K expression in both Smad4 and Smad1/5 Ad-CRE osteoclasts, and changes in c-fos and Nfatc1 expression in only Smad4 Ad-CRE cells. Lastly we also detected a significant decrease in resorption pits and area resorbed in both the Smad4 and Smad1/5 Ad-CRE osteoclasts. Because we inhibited osteoclast differentiation with loss of either Smad4 or Smad1/5 expression, we assessed whether BMPs affected osteoclast activity in addition to BMP's effects on differentiation. Therefore, we treated mature osteoclasts with BMP2 or with dorsomorphin, a chemical inhibitor that selectively suppresses canonical BMP signaling. We demonstrated that BMP2 stimulated resorption in mature osteoclasts whereas treatment with dorsomorphin blocks osteoclast resorption. These results indicate that the BMP canonical signaling pathway is important for osteoclast differentiation and activity.