Loss Of Smad4 Research Articles

Targeted Sequencing of Sorted Esophageal Adenocarcinoma Cells Unveils Known and Novel Mutations in the Separated Subpopulations.

INTRODUCTION:Our study aimed at investigating tumor heterogeneity in esophageal adenocarcinoma (EAC) cells regarding clinical outcomes.METHODS:Thirty-eight surgical EAC cases who underwent gastroesophageal resection with lymph node dissection in 3 university centers were included. Archival material was analyzed via high-throughput cell sorting technology and targeted sequencing of 63 cancer-related genes. Low-pass sequencing and immunohistochemistry (IHC) were used to validate the results.ResultsThirty-five of 38 EACs carried at least one somatic mutation that was absent in the stromal cells; 73.7%, 10.5%, and 10.5% carried mutations in tumor protein 53, cyclin dependent kinase inhibitor 2A, and SMAD family member 4, respectively. In addition, 2 novel mutations were found for hepatocyte nuclear factor-1 alpha in 2 of 38 cases. Tumor protein 53 gene abnormalities were more informative than p53 IHC. Conversely, loss of SMAD4 was more frequently noted with IHC (53%) and was associated with a higher recurrence rate (P = 0.015). Only through cell sorting we were able to detect the presence of hyperdiploid and pseudodiploid subclones in 7 EACs that exhibited different mutational loads and/or additional copy number amplifications, indicating the high genetic heterogeneity of these cancers.DISCUSSION:Selective cell sorting allowed the characterization of multiple molecular defects in EAC subclones that were missed in a significant number of cases when whole-tumor samples were analyzed. Therefore, this approach can reveal subtle differences in cancer cell subpopulations. Future studies are required to investigate whether these subclones are responsible for treatment response and disease recurrence.

Abstract 6161: PARP inhibition enhances radiotherapy of SMAD4 deficient human head and neck squamous cell carcinomas in experimental models

Abstract Purpose: SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study if SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). Experimental Design: We analyzed HNSCC TCGA data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a Phase I trial investigating olaparib were analyzed. Results: SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4 mutant but not SMAD4 positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4 deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared to vehicle treated tumors. In vitro, olaparib or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death and less proliferation in SMAD4 deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a Phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. Conclusion: SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs. Citation Format: Christian D. Young, Ariel L. Hernandez, Li Bian, Sana D. Karam, David Raben, Xiao-Jing Wang. PARP inhibition enhances radiotherapy of SMAD4 deficient human head and neck squamous cell carcinomas in experimental models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6161.

Abstract 5173: Identification of new targets in SMAD4-deficient cancers by whole genome CRISPR screening

Abstract The TGF-β signaling mediator SMAD4 is frequently mutated or deleted in colorectal cancer (CRC) and pancreatic cancers (PC) and is associated with poorer prognosis. This makes SMAD4 loss interesting to harness for synthetic lethality-based therapy. In this study, we first restored SMAD4 in SMAD4-deficient cell lines (HT29 and SW620) and showed that SMAD4 restoration reestablished the TGF-β-induced growth inhibition in vitro and in vivo, thereby confirming the tumor suppressive role of SMAD4 in these cell lines. In order to identify genetic vulnerabilities of SMAD4-deficient cells, we conducted pooled genome-wide loss-of-function CRISPR/Cas9 screens across four non-isogenic cell lines harboring altered (HT29 and SW620) or wild-type (DLD1 and MIAPACA2) SMAD4. We identified candidate genes, including the Krüppel-like factor 5 (KLF5), which has already been suggested as a potentially interesting target for drug therapy in SMAD4-deficient cancers. Novel candidates that might be interesting synthetic lethal targets in SMAD4-negative cancers will be presented. Citation Format: Christophe Marcireau, Hélène Erasimus, Vanessa Kolnik, Frederic Lacroix, Stéphane D'agostino, Olivier Lemaitre, Alexandre Rohaut, Isabelle Sanchez, Sukhvinder Sidhu, Gilbert Thill, Michel Didier, Laurent Debussche. Identification of new targets in SMAD4-deficient cancers by whole genome CRISPR screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5173.

Pattern of Invasion in Human Pancreatic Cancer Organoids Is Associated with Loss of SMAD4 and Clinical Outcome.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by extensive local invasion and systemic spread. In this study, we employed a three-dimensional organoid model of human pancreatic cancer to characterize the molecular alterations critical for invasion. Time-lapse microscopy was used to observe invasion in organoids from 25 surgically resected human PDAC samples in collagen I. Subsequent lentiviral modification and small-molecule inhibitors were used to investigate the molecular programs underlying invasion in PDAC organoids. When cultured in collagen I, PDAC organoids exhibited two distinct, morphologically defined invasive phenotypes, mesenchymal and collective. Each individual PDAC gave rise to organoids with a predominant phenotype, and PDAC that generated organoids with predominantly mesenchymal invasion showed a worse prognosis. Collective invasion predominated in organoids from cancers with somatic mutations in the driver gene SMAD4 (or its signaling partner TGFBR2). Reexpression of SMAD4 abrogated the collective invasion phenotype in SMAD4-mutant PDAC organoids, indicating that SMAD4 loss is required for collective invasion in PDAC organoids. Surprisingly, invasion in passaged SMAD4-mutant PDAC organoids required exogenous TGFβ, suggesting that invasion in SMAD4-mutant organoids is mediated through noncanonical TGFβ signaling. The Rho-like GTPases RAC1 and CDC42 acted as potential mediators of TGFβ-stimulated invasion in SMAD4-mutant PDAC organoids, as inhibition of these GTPases suppressed collective invasion in our model. These data suggest that PDAC utilizes different invasion programs depending on SMAD4 status, with collective invasion uniquely present in PDAC with SMAD4 loss. SIGNIFICANCE: Organoid models of PDAC highlight the importance of SMAD4 loss in invasion, demonstrating that invasion programs in SMAD4-mutant and SMAD4 wild-type tumors are different in both morphology and molecular mechanism.

PARP Inhibition Enhances Radiotherapy of SMAD4-Deficient Human Head and Neck Squamous Cell Carcinomas in Experimental Models.

SMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT). We analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed. SMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage. SMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.

Abstract B11: Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila

Abstract Colorectal cancer (CRC) is an extremely complex and genetically diverse disease with few available models that recapitulate the key features of heterogeneity. The Cancer Genome Atlas has identified 30 recurrent mutated genes as drivers of CRC. Individual tumors can carry any number of combinations of these mutations. A critical challenge in research has been the ability to model these genetically heterogenous tumors in animal systems. Highly sophisticated genetic tools in Drosophila make it an ideal model system to create and study different genome-based CRC tumors that well complement other animal models. Highly conserved signaling pathways and high similarity in gut homeostasis also make it a powerful and representative system to better understand CRC tumors and their progression. We have previously generated a panel of genetically complex Drosophila models that reflect the genomic landscape of sequenced colon tumors. The most complex model in this panel carried transgenic constructs to knock down Drosophila orthologs of tumor suppressors TP53, APC, SMAD4, and PTEN as well as to express oncogenic KRASG12V. However, creating more complex models using standard Drosophila crosses has been challenging. To address this, we have generated a multigenic vector that carries three different multiple cloning sites (MCSs) designed for cloning three separate transgenes. Two are designed for inducible protein expression to model oncogenic activation while the third is for a micro-RNA inspired synthetic multi-hairpin cluster to knock down multiple tumor suppressors. Here, we will report our efforts to further validate and expand this technology to build more complex cancer models. Using a series of test constructs, we show that transgenic flies carrying synthetic clusters of as many as 16 hairpins can be generated. Hairpins expressed in the context of synthetic clusters are as effective as single hairpins with no detectable positional effects. Finally, by introducing additional design elements such as intron-mediated hairpin cluster expression and bicistronic protein expression using a T2A self-cleaving peptide design, we have generated transgenes that express two proteins and up to 16 hairpins from a single transcript. We then used this updated platform to build a proof-of-concept colorectal cancer model that reflects IRS2 amplification, oncogenic KRASG12V, and loss of tumor suppressors TP53, APC, SMAD4, and SMAD2 from a single transcript designed for inducible expression. This new design frees up the remaining two cloning sites in our multigenic vector for additional genetic manipulations and pushes the limits of our technology to build a new generation of colorectal cancer models to study features of human tumors that have been traditionally challenging to capture in experimental models. Using this technology, we hope to better understand tumor development and find potential therapies that can specifically target these unique, personalized CRC genome-based models. Citation Format: Maria Quintero, Alexander Teague, Ross Cagan, Erdem Bangi. Use of novel multigenic vectors to model complex genome-based colorectal cancer profiles in Drosophila [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B11.

Smad4-dependent morphogenic signals control the maturation and axonal targeting of basal vomeronasal sensory neurons to the accessory olfactory bulb.

ABSTRACTThe vomeronasal organ (VNO) contains two main types of vomeronasal sensory neurons (VSNs) that express distinct vomeronasal receptor (VR) genes and localize to specific regions of the neuroepithelium. Morphogenic signals are crucial in defining neuronal identity and network formation; however, if and what signals control maturation and homeostasis of VSNs is largely unexplored. Here, we found transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) signal transduction in postnatal mice, with BMP signaling being restricted to basal VSNs and at the marginal zones of the VNO: the site of neurogenesis. Using different Smad4 conditional knockout mouse models, we disrupted canonical TGFβ/BMP signaling in either maturing basal VSNs (bVSNs) or all mature VSNs. Smad4 loss of function in immature bVSNs compromises dendritic knob formation, pheromone induced activation, correct glomeruli formation in the accessory olfactory bulb (AOB) and survival. However, Smad4 loss of function in all mature VSNs only compromises correct glomeruli formation in the posterior AOB. Our results indicate that Smad4-mediated signaling drives the functional maturation and connectivity of basal VSNs.

Association of SMAD4 loss with response to neoadjuvant chemotherapy with the autophagy inhibitor hydroxychloroquine in patients with pancreatic adenocarcinoma.

761 Background: SMAD4, a tumor suppressor gene, is inactivated or deleted in 60-90% of pancreatic adenocarcinomas (PDA). Loss of SMAD4 allows tumor progression by limiting cell cycle arrest and apoptosis and increasing metastases. SMAD4 deficient PDA cells are resistant to radiotherapy by upregulation of autophagy, a cell survival mechanism that allows intracellular recycling of macromolecules and organelles. Hydroxychloroquine (HCQ) is a known autophagy inhibitor, suggesting that HCQ treatment in SMAD4 deficient PDA may prevent therapeutic resistance induced by autophagy upregulation. Methods: We retrospectively analyzed the SMAD4 status of PDA patients enrolled in two prospective clinical trials evaluating preoperative HCQ. The first dose escalation trial demonstrated the safety of preoperative gemcitabine with HCQ ( NCT01128296 ). More recently, a randomized trial of gemcitabine/nab-paclitaxel +/- HCQ evaluated Evans Grade histopathologic response ( NCT01978184 ). Immunohistochemistry of resected specimens for SMAD4 was previously performed. Patients not treated at the max HCQ dose (n = 5), not resected (n = 2) or with SMAD4 staining unavailable were excluded (n = 10). The effect of SMAD4 loss on response to HCQ and chemotherapy was studied for association with clinical outcome. Fisher’s exact test and log-rank test were used to assess response and survival. Results: 52 patients receiving HCQ with neoadjuvant chemotherapy and 24 patients receiving neoadjuvant chemotherapy alone were studied. Of the HCQ group, 25 patients had SMAD4 loss (48%), compared with 15 control patients (63%, p = 0.32). 76% of HCQ treated patients with SMAD4 loss obtained a histopathologic response ≥2A, compared to only 37% with SMAD4 intact (p = 0.006). In the control group, loss of SMAD4 was associated with a nonsignificant detriment in 3 year OS (25% vs. 78%, p = 0.3) that was less apparent in patients treated with HCQ (46% vs. 47%, p = 0.18). Conclusions: The addition of HCQ to neoadjuvant chemotherapy in PDA may improve treatment response in patients with SMAD4 loss. Further study of the relationship between SMAD4, autophagy and treatment outcomes in PDA is warranted.

Abstract C40: Dissecting key biologic processes in pancreatic cancer metastasis using a genetically defined 3D organoid model

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, with a dismal 5-year survival rate of 6-8% due to its late detection and limited response to treatment (1,2). Multiple combinations of genetic alterations are frequently found in human PDAC, with KRAS activating mutation representing the main driver, followed by loss of tumor-suppressor genes CDKN2A, TP53, and SMAD4 as the disease progresses (3). 90% of PDAC patients die of metastatic disease (1); however, the cellular and molecular mechanisms involved in establishing the metastatic disease are understudied compared to those involved in tumor initiation. Owing to this, improved biologic understanding of PDAC metastasis remains warranted in order to develop effective therapeutic regimens for this disease. 3D organoid cultures derived from human and mouse PDAC tumors have recently become a model of choice to study PDAC tumorigenesis, gradually replacing more commonly used 2D cancer cell lines. Organoids maintain similar gene expression profiles as the in vivo situation when cultured in a 3D matrix, and genetic manipulation is feasible. We generated a panel of isogenic murine pancreatic intraepithelial neoplasia (PanIN) and PDAC organoids containing genetic mutations commonly found in human PDAC (KRAS, TP53, SMAD4, CDKN2A), either alone or in combination, using CRISPR-Cas9 technology. Since large-scale in vitro production of these PDAC organoids is achievable, the platform can be used for a multitude of downstream analyses in vitro and in vivo, such as drug screening and metastasis-related studies. The parental of these organoids is isolated from C57/Bl6 mice, which can be orthotopically transplanted into immunocompetent mice, and the resulting tumor recapitulates human and GEMM PDAC development. In the process of systematic characterization of these organoid lines, we found that organoids with more mutations exhibit higher proliferation rates in vitro and tumor formation rates in vivo. These organoids also showed genotype-dependent organotropism during metastatic colonization. We further used this unique tool to systematically address if each combination of tumor suppressor loss utilizes distinct molecular mechanisms to drive metastasis initiation and invasion. This will allow us to identify druggable pathways that could inhibit metastasis initiation/invasion and progression. Thus, these organoids provide a powerful platform for disease modeling and drug discovery both in vitro and in vivo. Citation Format: Fong Cheng Pan, Angie Ai-Nok Chong, Steven D. Leach, Shuibing Chen, Todd Evans. Dissecting key biologic processes in pancreatic cancer metastasis using a genetically defined 3D organoid model [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C40.

P118 LOSS OF SMAD4 EXPRESSION IN ESOPHAGEAL ADENOCARCINOMA (EAC) IS CORRELATED TO POOR SURVIVAL

Abstract Aim Our study is aimed to evaluate the impact of SMAD4 expression on the clinical outcome of therapy for esophageal adenocarcinoma (EAC). Background & Methods Esophageal adenocarcinoma is characterized by a high genetic heterogeneity and a low survival rate, despite the adoption of aggressive therapies. We evaluated the expression of SMAD4, a tumor suppressor gene frequently mutated in cancer. by immunohistochemistry in 67 formalin-embedded (FFPE) EAC surgical specimens (patients were primarily treated with surgical resection-naïve cases), 34 of which were analyzed for SMAD4 gene by Next Generation Sequencing (NGS) with a dedicated target panel. Loss of SMAD4 protein was defined by a complete loss of expression in least 30% of cancer cells. Survival curves were determined using Kaplan–Meier methods. Results Loss of SMAD4 immunoreactivity was found in 33 out of 67 EAC cases (49.3%). Among a subset of 34 cases assessed in NGS, SMAD4 mutations were found in 3 cases (8.8%), all associated with protein loss. Loss of SMAD4 expression was also found in several cases with no mutations in SMAD4 gene (17/34, 50%). Loss of SMAD4 immunoreactivity was associated with poor overall survival (p=0.013) and higher risk of recurrence (p=0.001). Conclusion Loss of SMAD4 expression was a recurrent event in EAC, linked with genetic mutations in few cases, whereas in the majority of cases it might be related to epigenetic mechanisms, such as promoter hypermethylation. SMAD4 loss was strongly associated with recurrence and poor survival in patients, suggesting SMAD4 expression as potential prognostic biomarker in EAC. Further studies are required to develop strategic therapeutic options directly targeting SMAD4 and/or its regulators.

Transforming Growth Factor-β Signaling Pathway in Colorectal Cancer and Its Tumor Microenvironment.

Transforming growth factor-beta (TGF-β) signaling is one of the important cellular pathways that play key roles for tissue maintenance. In particular, it is important in the context of inflammation and tumorigenesis by modulating cell growth, differentiation, apoptosis, and homeostasis. TGF-β receptor type 2 (TGFBR2) mutations affected by a mismatch repair deficiency causes colorectal cancers (CRCs) with microsatellite instability, which is, however, associated with relatively better survival rates. On the other hand, loss of SMAD4, a transcription factor in the TGF-β superfamily signaling, promotes tumor progression. Loss of heterozygosity on chromosome 18 can case SMAD4-deficient CRC, which results in poorer patients’ survival. Such bidirectional phenomenon driven by TGF-β signaling insufficiency reflects the complexity of this signaling pathway in CRC. Moreover, recent understanding of CRC at the molecular level (consensus molecular subtype classification) provides deep insight into the important roles of TGF-β signaling in the tumor microenvironment. Here we focus on the TGF-β signaling in CRC and its interaction with the tumor microenvironment. We summarize the molecular mechanisms of CRC tumorigenesis and progression caused by disruption of TGF-β signaling by cancer epithelial cells and host stromal cells.

Localisation of PGK1 determines metabolic phenotype to balance metastasis and proliferation in patients with SMAD4-negative pancreatic cancer

ObjectivePancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of GI tumour, and it possesses deregulated cellular energetics. Although recent advances in PDAC biology have led to the discovery of...

Expression of SMAD4 is Retained in Most Gynecologic Tumors with Mucinous Differentiation.

SMAD4 is a tumor suppressor gene that plays a role in cancer initiation and progression. A few studies have explored the value of immunohistochemistry for SMAD4 in gynecologic neoplasms, mainly in the ovary. However, literature is sparse when it comes to other sites such as endometrium and cervix, as well as in benign and borderline ovarian mucinous neoplasms. The aim of this study was to assess the expression of SMAD4 in various gynecologic tumors. We selected primary gynecologic tumors comprising a spectrum of neoplasms showing mucinous differentiation. Few cases of metastatic tumors were also included. A total of 103 cases were retrieved, including tumors of ovarian origin (13 mucinous adenocarcinomas, 9 mucinous borderline tumors, 19 mucinous cystadenomas, and 3 mucinous tumors arising from teratomas), 36 of endometrial origin (23 endometrioid adenocarcinomas with mucinous differentiation and 13 mucinous adenocarcinomas), 17 cases of cervical carcinoma (16 of usual type and 1 of gastric type), and 6 metastatic adenocarcinomas to ovary. SMAD4 immunohistochemistry was retained in most primary tumors, except in 3 endocervical adenocarcinomas (2 usual-type, 1 gastric-type) and in one mucinous carcinoma arising from an ovarian teratoma. Of the 6 metastatic cases, 4 showed SMAD4 loss. In summary, retained expression of SMAD4 was seen in 95.8% of primary gynecologic neoplasms. These results can be of utility when dealing with mucinous lesions for which metastatic origin is suspected. Loss of SMAD4 expression virtually excludes primary tumors of endometrial or ovarian origin, but is of less utility when evaluating carcinomas involving the cervix.

Immunohistochemical profiling of liver metastases and matched-pair analysis in patients with metastatic pancreatic ductal adenocarcinoma

BackgroundThe purpose of the current study was to investigate the immunohistochemical (IHC) profile of liver metastases (LM) in patients with pancreatic ductal adenocarcinoma (PDAC). MethodsExpression of 15 IHC markers in liver biopsies from 77 patients with PDAC, who were diagnosed between 2010 and 2014, were evaluated. In a separate subgroup analysis (n = 12), paired samples (LM and primary tumor) from the same patient were investigated for IHC profile differences. ResultsLM samples were classified as pancreatobiliary-type (PB-type) in 72 patients (93.5%), intestinal-type (INT-type) in four patients (5.2%), and squamous in one patient (1.3%). There was no significant difference in overall survival (OS) between LM of the PB-type or INT-type (p = 0.097). In a multivariate analysis, age <70 years (p = 0.047), absence of SMAD4 mutation (p = 0.026), absence of CDX2 expression (p = 0.003), and well to moderate differentiation were significant prognostic factors for better OS in patients with LM (p = 0.031). Analysis of paired tissue samples from LM and the primary tumor revealed a difference in CDX2 (50% increase, p = 0.125) and SMAD4 (33% loss of SMAD4, p = 0.375). ConclusionsCDX2 expression and SMAD4 mutation indicate a poor outcome in patients with LM of PDAC. Matched-pair analysis revealed differences in distinct IHC marker expression.

Loss of SMAD4 Expression Associated with Worse Progression-Free and Overall Survival in Patients with Pancreatic Adenocarcinoma Treated Surgically

The tumor suppressor gene SMAD4 is inactive in roughly half of patients with pancreatic adenocarcinoma. Several studies have linked los23s of SMAD4 with worse oncologic outcomes, while other studies have refuted this association. In this study, we examined the association of SMAD4 loss with oncologic outcomes in a cohort of patients with pancreatic adenocarcinoma treated with resection +/- adjuvant chemo-radiotherapy. Patients with non-metastatic adenocarcinoma of the pancreas s/p radical resection +/- adjuvant chemo-radiotherapy at the University of Pennsylvania were included. SMAD4 status was determined by immunolabeling assessment from the surgical pathology. Multiple assessments of SMAD4 status were obtained for each patient and the maximum, mean, and median value were analyzed. Kaplan-Meier survival analysis and Cox regression analysis was performed to assess the association of SMAD4 status with different oncologic outcomes of interest [(local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), progression-free survival (PFS), and overall survival, (OS)]. PFS was defined as time to first recurrence or death. P<0.05 was considered significant. 66 patients had evaluable SMAD4 tumor status and were included in the analysis. 59 received adjuvant chemo-radiotherapy; 7 received adjuvant chemotherapy alone. Median age was 65 (range 43 – 85). 35 patients were male (53%). Median follow-up was 23.5 months (range 3 – 141 months). 40 of 66 patients had complete loss of SMAD4 (61%). There were no significant differences in baseline demographic, clinical, pathologic, and treatment-related variables between the patients with intact vs. lost SMAD4 (p>0.05). On univariable Cox regression analysis, SMAD4 intact status was marginally associated with improved DMFS [HR 0.74 (95%CI 0.54-1.01), p=0.06) but was not predictive of LRFS [HR 0.96 (95%CI 0.67-1.39), p=0.85]. SMAD4 intact status was associated with significantly improved PFS [HR 0.72 (95%CI 0.54-0.95), p=0.02] and overall survival [HR 0.73 (95%CI 0.55-0.98), p=0.04). Median survival for patients with intact SMAD4 vs. SMAD4 loss was 37 months vs. 21 months (log-rank p=0.08). Results were similar when analyzed using median and mean SMAD4 values. For patients treated with surgical resection +/- adjuvant chemo-radiotherapy, loss of SMAD4 expression correlated with significantly worse progression-free survival and overall survival compared to patients with intact SMAD4. Loss of SMAD4 may help to predict patients at higher risk for recurrence following surgical resection and could help to guide future adjuvant therapy or help select patients for surgery or definitive radiotherapy.

Dysregulated Tgfbr2/ERK-Smad4/SOX2 Signaling Promotes Lung Squamous Cell Carcinoma Formation.

Lung squamous cell carcinoma (SCC) is a common type of lung cancer. There is limited information on the genes and pathways that initiate lung SCC. Here, we report that loss of TGFβ type II receptor (Tgfbr2), frequently deleted in human lung cancer, led to predominant lung SCC development in KrasG12D mice with a short latency, high penetrance, and extensive metastases. Tgfbr2-loss-driven lung SCCs resembled the salient features of human lung SCC, including histopathology, inflammatory microenvironment, and biomarker expression. Surprisingly, loss of Smad4, a key mediator of Tgfbr2, failed to drive lung SCC; instead, low levels of phosphorylated ERK1/2, a Smad-independent downstream effector of Tgfbr2, were tightly associated with lung SCC in both mouse and human. Mechanistically, inhibition of phosphorylated ERK1/2 significantly upregulated the expression of SOX2, an oncogenic driver of lung SCC, and cooperated with SMAD4 repression to elevate SOX2. Inhibition of ERK1/2 in Smad4fl/fl ;KrasG12D mice led to extensive lung SCC formation that resembled the SCC phenotype of Tgfbr2-deficient mice. Overall, we reveal a key role of ERK1/2 in suppressing SCC formation and demonstrate that dysregulated Tgfbr2/ERK-Smad4/SOX2 signaling drives lung SCC formation. We also present a mouse model of metastatic lung SCC that may be valuable for screening therapeutic targets. SIGNIFICANCE: This study sheds new light on the mechanisms underlying lung SCC formation driven by mutated Kras.

Loss of SMAD4 protein expression in gastrointestinal and extra-gastrointestinal carcinomas.

SMAD4 (DPC4) is a tumour suppressor gene that is dysregulated in various tumour types, particularly pancreaticobiliary and gastrointestinal carcinomas. Corresponding loss of protein expression has been reported in approximately 50% of pancreatic and 25% of colonic adenocarcinomas. In the evaluation of carcinoma of unknown primary site, immunohistochemical loss of SMAD4 expression is often used to suggest pancreaticobiliary origin, but there are limited data on the spectrum of SMAD4 expression in carcinomas of other sites. This study evaluates the frequency of SMAD4 loss in a large cohort of carcinomas from diverse anatomical sites. Immunohistochemistry for SMAD4 was performed on tissue microarrays or whole tissue sections of 1210 carcinomas from various organs: gastrointestinal tract, liver, pancreas/biliary tract, lung, breast, thyroid, kidney, ovary and uterus. Expression was considered lost when there was complete absence of staining in tumour cell nuclei, in the presence of intact staining in non-neoplastic cells. SMAD4 loss was seen in 58% of pancreatic adenocarcinomas, 27% of appendiceal adenocarcinomas, 19% of colorectal adenocarcinomas, 16% of cholangiocarcinomas, 10% of lung adenocarcinomas and <5% of oesophageal, breast, gastric and mucinous ovarian adenocarcinomas. All papillary thyroid, hepatocellular, non-mucinous ovarian, endometrial and renal cell carcinomas showed intact SMAD4 nuclear expression. In addition to pancreaticobiliary, appendiceal and colonic tumours, SMAD4 loss is also seen in a small subset of other carcinomas, specifically breast, lung, oesophageal and gastric adenocarcinomas, all of which are typically CK7-positive, similar to pancreaticobiliary carcinoma. Awareness of SMAD4 loss in these other carcinoma types is helpful in the evaluation of carcinomas of unknownor uncertain primary site.

Abstract 5314: Smad4 loss upregulates chemokine expression and promotes tumor inflammation and growth in head and neck cancer

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide. Of the patients diagnosed with HNSCC, previous findings show that SMAD4 loss is more common in recurrent HNSCC patients compared with newly diagnosed cases and is strongly associated with poor patient survival and metastases. SMAD4 is a tumor suppressor gene and is a central mediator of the transforming growth factor β (TGF-β) signaling pathway and acts as a co-SMAD which binds receptor-regulated SMADs such as SMAD2 and 3. SMAD2/3/4 complex is important for regulating downstream gene transcription that is involved in tumor invasion and metastasis. Purpose: Since the TGF-β pathway is highly dysregulated in HNSCC, we investigate in this study how SMAD4 downregulation alters TGF-β pathway signaling and the tumor microenvironment. Results: Using isogenic HNSCC cell lines, we visualized using immunofluorescence staining that levels of activated phospho-SMAD2 in the nucleus are correlated with expression of SMAD4. Genome-wide screening using Gene Set Enrichment Analysis (GSEA) further revealed changes in gene expression during SMAD4 knockdown in HNSCC cells. From GSEA results, the 20 most significantly altered Gene Ontology (GO) pathways were determined. It was shown that majority of these GO pathways are associated with regulating neutrophil migration and chemotaxis with an upregulation of CXC chemokine gene expression, specifically with CXCL1. In contrast, anti-tumor chemokines, such as CXCL9 and CXCL11, were downregulated during loss of SMAD4 expression. Using an orthotopic mouse model we show that in vivo, decreased expression of SMAD4 promotes tumor take and growth. Conclusion: Collectively, these results suggest that HNSCC tumors with SMAD4 loss may have poor prognosis attributed to immunosuppressive functions of chemokine expression that acts on tumor-associated neutrophils. Citation Format: Jude Masannat, Xuefeng Wang, Biwei Cao, FeiFei Song, Ritu Chaudhary, Janis de la Iglasia, Ciaara Gorgoglione, Robbert Slebos, Christine Chung. Smad4 loss upregulates chemokine expression and promotes tumor inflammation and growth in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5314.

Abstract 3216: CKD-516, a novel vascular disrupting agent, enhances anticancer activity of anti-PD-1 antibody in SMAD4-deficient colon cancer model

Abstract CKD-516 is a potent vascular disrupting agent (VDA), selectively acting on tumor vessels. A clinical study of CKD-516 combined with irinotecan is undergoing in colorectal cancer. There have been exponential gains in immune-oncology (I-O) in recent times through the development of immune checkpoint inhibitors (ICIs). ICIs demonstrated durable response and some patients achieved disease control for several years. However, there are still critical unmet medical needs for the combination therapies due to limited response rate of ICIs. The first step of the cancer-immunity cycle is dendritic cells (DCs) maturation which is necessary for the anticancer immunity. It is well known that mature DCs play critical roles in priming immune responses in cancer patients. We found that CKD-516 is able to induce DC maturation through Rho signaling pathway in DCs. A tumor suppressor gene, SMAD4 is associated with several cancers including stomach cancer, colorectal cancer, and pancreas cancer. The loss of SMAD4 is a poor prognostic biomarker in these cancer types and its mutation is related to poor clinical outcome on immunotherapies. A SMAD4-deficient mouse colorectal cancer cell line (3349LM) was primary cultured from a spontaneous intestinal adenocarcinoma formed in a Villin-Cre;Smad4(F/F);Trp53(F/F) mouse. CKD-516 showed a synergistic effect with anti-PD-1 antibody in 3349LM syngeneic model. In addition, 3349LM is also a MSS type cancer. Thus, CKD-516 displayed a therapeutic potential for several cancer patients who do not respond to immunotherapy. In summary, CKD-516 is a novel VDA with immune boosting effect. In SMAD4 deficient cancer model, CKD-516 has shown synergistic effects in combination with PD-1 antibody. Therefore, these data suggest that CKD-516 potentiates the anticancer activity of immunotherapy. Citation Format: Soo Jin Kim, Hark Kyun Kim, Keun Ho Ryu, Chung Il Hong. CKD-516, a novel vascular disrupting agent, enhances anticancer activity of anti-PD-1 antibody in SMAD4-deficient colon cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3216.

Abstract 4632: A new mouse model for rapid identification of key factors driving prostate cancer progression and invasiveness

Abstract Prostate Cancer is amongst the most frequently diagnosed malignancies and the second leading cause of cancer-related death in men worldwide accompanied by an increasing incidence. Until now its heterogeneity has been a major challenge in the establishment of good in vivo models for fast validation of potential driver genes. Classic in vivo models are costly, time-consuming and often target the majority of the prostate epithelium. We successfully introduced a novel prostate cancer mouse model based on CRISPR/Cas9 technology, ensuring multiplexed gene editing. In this model, specific in vivo gene editing was obtained in murine prostate epithelium cells of a transgenic mouse strain, harboring the CRISPR associated protein 9 (Cas9) endonuclease. Prostate epithelium cells were transduced by an Adeno-associated virus (AAV), carrying multiple single guide RNAs (sgRNA). Genetically different viral constructs were designed, each expressing different sgRNA combinations against Pten, representing the main driver in prostate cancer, tumor suppressor Trp53 and either one of the AP1 transcription factor subunits Junb or Fos, or tumor suppressor Smad4. Since viral transduction occurs in only a few cells, edited cell clones can clonally expand and undergo a natural selection process. Furthermore, the simultaneous gene knockouts reflect the human scenario of tumor heterogeneity. Mouse prostates were isolated and analyzed three to nine months post-injection to obtain insight on whether or not a gene appears crucial for tumor development in a specific tissue and wherein different gene combinations correlate with tumor severity. Histological analysis revealed increased proliferation, increased AKT activation, as well as invasiveness in samples with multiple gene knockouts compared to controls with single Pten knockout. Furthermore, loss of SMAD4 accelerated cancer progression when compared to the loss of the AP-1 transcription factor. Surprisingly, knockout of Trp53 was rarely observed in prostate samples while it occurred frequently in other tumor types in the same model system. This indicates that knockout of Trp53 may not be required for prostate cancer initiation. Overall, we established an in vivo model system of simultaneous, multiplexed gene editing in the prostate epithelium to address the biological cross-talk between various, altered pathways in prostate cancer initiation and progression. Citation Format: Maria Riedel, Latifa Bakiri, Martin F. Berthelsen, Michael Borre, Mikkel H. Vendelbo, Erwin F. Wagner, Martin K. Thomsen. A new mouse model for rapid identification of key factors driving prostate cancer progression and invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4632.