ConclusionSMAD4 is a critical tumor suppressor in cancers (Zhang, et al., 2010). Thus, it is imperative to understand which tumorigenic pathways are regulated in a SMAD4-dependent manner. Our studies focus on whether SMAD4 suppresses invasive properties of serrated cancers. Our results show that tumor organoids derived from our SMAD4KO; BrafV600E/+ mouse model exhibit invasive characteristics, suggesting that loss of SMAD4 accelerates serrated tumor invasiveness (Figure 1). By re-expressing SMAD4 in our tumor organoids using a pINDUCER system, we have generated a model to directly test whether SMAD4 suppresses cancer invasion. Our results show that SMAD4 expression not only reduces the invasive phenotype, but it also leads to downregulation of genes involved in invadopodia formation (CTTN, MMP14, IL6, CXCL3) and angiogenesis (PDGFA and VEGFA) (Figures 2 and 3). These findings suggest that SMAD4 works antagonistically to pathways that lead to cancer invasion.

Investigating the Role of SMAD4 in Serrated Colorectal Cancer Using an Inducible Organoid System

Manisha Bandari, Jillian Carrick, Katherine Haro, Dr. Kevin Tong, Dr. Michael VerziHuman Genetics Institute of New Jersey

AbstractColorectal cancer (CRC) is one of the most common cancers in the United States. A small portion of CRCs progress though the more aggressive serrated tumor pathway, as opposed to the canonical “adenoma” pathway. While the SMAD4/TGFβ pathway is mutated in 57% of CRCs (Cancer Genome Atlas Network, 2012), the role of SMAD4 in the serrated pathway has not yet been established. The use of an organoid system provides the capability to re-express SMAD4 and directly test how SMAD4 re-expression impacts serrated tumors using a Doxycycline construct. My studies seek to address if re-expression of SMAD4 in tumor organoids will reduce the invasive phenotype normally seen in tumor organoids and decrease expression of genes involved in invasion. We indeed did see a reduction of the metastatic phenotype in SMAD4KO; BRAFV600E/+ tumor organoids and a downregulation in genes associated with colorectal cancer invasion.

BackgroundColorectal cancer (CRC) is the 3rd most common cancer in the United States (Siegel, et al., 2017). The “serrated” CRC pathway, unlike the canonical WNT-driven adenoma pathway, is driven by oncogenic mutations in the BRAF gene. This leads to amplified MAPK signaling and causes overexpression of epithelial growth factors and is commonly associated with the initiation of CRC (Tsai, 2015) (Stefanius, 2011) (Fang, Ricardson, 2005) (Tong, et al., 2017) (Kambara, 2004). The SMAD4/TGFβ pathway is mutated in 57% of CRCs (Cancer Genome Atlas Network, 2012). The role of SMAD4 in the serrated tumor pathway has not yet been established. However, loss of SMAD4 in a BRAFV600E/+ mutant mouse accelerates the formation of tumors (Tong, et al., 2017). This project will use a murine intestinal organoid system to mimic the intestinal tract. The Verzi lab has previously seen SMAD4KO; BRAFV600E/+ tumor organoids exhibit an invasive behavior. This behavior shows organoids growing projections in the direction of nearby organoids for the purpose of connecting with them. This phenotype is a hallmark of invasive cancer cells (Sibony-Benyamini, Gil-Henn., 2012). SMAD4 is documented to be a key player in suppressing cancer invasion and metastasis (Zhang, et al., 2010). A pINDUCER-SMAD4 plasmid system will be used to determine the phenotypic effects of SMAD4 re-expression in invasive tumor organoids.

Future DirectionsGiven that these processes are downregulated in the presence of SMAD4, we will seek to address whether the activation of the invadopodia or angiogenic pathways are critical for invasion of cancer cells. Using CTTN as an example, one could culture SMAD4KO; BrafV600E/+ tumor organoids and transfect them with a CTTN-siRNA (Zhang, et al., 2016) which would inactivate CTTN expression in the organoids. Organoids will be seeded in 6 wells, with about 50 organoids per well. Three wells will be given a typical culture media, while the other three wells will be treated with CTTN-siRNA to suppress invadopodia gene CTTN and will be confirmed using qPCR. All organoids will be imaged, and the number of connections formed between organoids will be counted daily. Based on our current findings, we would expect that SMAD4KO; BrafV600E/+ tumor organoids would develop the invasive networks within 3 days. If CTTN is critical for the invasive function, then organoids treated with CTTN-siRNA would show a significant reduction in the invasive networking phenotype. This process would be replicated for other invadopodia or angiogenic genes. By determining which gene pathways are critical in the cancer metastasis, we will gain a better understanding of the important pathways to target with therapeutics for treatment of invasive colorectal cancer. Alternatively, by probing SMAD4 induced and un-induced tumor organoids for a protein of interest, we will be able to visualize how SMAD4 suppresses the invasive process. We would first fix SMAD4 induced and uninduced SMAD4KO; BrafV600E/+ tumor organoids in 4% paraformaldehyde overnight, paraffin embed, and then section (4-5 um) (Neal, et al., 2018). Sections will then be stained with the corresponding antibody for the protein target of interest; in the case of CTTN, anti-CTTN (Anti-Cortactin (p80/85) Antibody, clone 4F11; Millipore). Since CTTN shows a reduced gene expression in the presence of SMAD4, low to no visualization of the antibody on the stained induced organoid sections will further confirm that SMAD4 reduces invadopodia formation in our organoids. Additionally, we may see a change in localization of CTTN expression. These findings would also support our conclusion that SMAD4 expression results in a down-regulation of genes involved in invadopodia formation and angiogenesis.

AcknowledgementsI would like to thank Dr. Michael Verzi for letting me be a part of his wonderful lab and partake in the amazing research being done. I would also like to thank Dr. Kevin Tong for being a great mentor and teaching me everything I know. A big thank you to Jillian Carrick and Katherine Haro for supporting me and for being awesome team members. Lastly, I would like to give a special thanks to the Human Genetics Institute of New Jersey as well as Duncan and Nancy MacMillan for the opportunity to be able to conduct and present my research this summer.

References- Alazzouzi H, Alhopuro P, Salovaara R, Sammalkorpi H, Järvinen H, Mecklin J-P, Hemminki A, Schwartz S, Aaltonen LA, Arango D. SMAD4 as a prognostic marker in colorectal cancer. Clin Cancer Res. 2005;11(7):2606–11- Cancer Genome Atlas Network. "Comprehensive molecular characterization of human colon and rectal cancer." Nature 487.7407 (2012): 330.- Deckers, Martine, et al. "The tumor suppressor SMAD4 is required for transforming growth factor β–induced epithelial to mesenchymal transition and bone metastasis of breast cancer cells." Cancer research 66.4 (2006): 2202-2209.- Fang, J.Y., Richardson, B.C., 2005. The MAPK signalling pathways and colorectal cancer. Lancet Oncol. 6 (5).- Kambara, T., et al. "BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum." Gut 53.8 (2004): 1137-1144.- Miyaki, Michiko, and Toshio Kuroki. "Role of SMAD4 (DPC4) inactivation in human cancer." Biochemical and biophysical research communications 306.4 (2003): 799-804.- Neal, James T et al. “Organoid Modeling of the Tumor Immune Microenvironment.” Cell vol. 175,7 (2018): 1972-1988.e16. doi:10.1016/j.cell.2018.11.021- Sibony-Benyamini, Hadas, and Hava Gil-Henn. “Invadopodia: the leading force.” European journal of cell biology vol. 91,11-12 (2012): 896-901.- Siegel RL, Miller KD, Fedewa SA, et al. Colorectal cancer statistics, 2017. CA Cancer J Clin. 2017; 67: 177- 193- Stefanius, Karoliina, et al. "Frequent mutations of KRAS in addition to BRAF in colorectal serrated adenocarcinoma." Histopathology 58.5 (2011): 679-692.- Tsai, Jia-Huei, et al. "Aberrant expression of annexin A10 is closely related to gastric phenotype in serrated pathway to colorectal carcinoma." Modern pathology 28.2 (2015): 268.- Tong, Kevin et al. “Degree of Tissue Differentiation Dictates Susceptibility to BRAF-Driven Colorectal Cancer.” Cell reports vol. 21,13 (2017): 3833-3845. doi:10.1016/j.celrep.2017.11.104- Zhang, Bixiang, et al. "Antimetastatic role of SMAD4 signaling in colorectal cancer." Gastroenterology 138.3 (2010): 969-980.- Zhang, Xiaojian et al. “Cortactin promotes colorectal cancer cell proliferation by activating the EGFR-MAPK pathway.” Oncotarget vol. 8,1 (2017): 1541-1554.

Re-expression of SMAD4 Downregulates Invadopodia and Angiogenesis Genes

Loss of SMAD4 Accelerates Formation of an Invasive PhenotypeA Day 1 Day 4 Day 7

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Fig 1. (A) Organoid line WT control and SMAD4KO; BRAFV600E/+ adjacent normal organoids form no connections. SMAD4KO; BRAFV600E/+ organoids derived from tumors do exhibit this invasive phenotype. Examples of

connections made between organoids are indicated by the arrows. 4x images on Day 1, 4, and 7 are shown. The scale bar is equivalent to 0.5 millimeters. Images are representative of four technical replicates. Images are representative of three biological replicates for SMAD4KO; BRAFV600E/+ tumor and adjacent normal organoids and one biological replicate for WT organoids.

(B) Quantitative counts of organoid connections were normalized to the number of viable organoids in the well. Counts were done on days 1, 4, and 7. Values are representative of four technical replicates. Values are representative of three biological replicates for SMAD4KO; BRAFV600E/+ tumor and adjacent normal organoids and one biological replicate for WT organoids.

Fig 2.(A) Tumor organoid line SMAD4KO; BRAFV600E/+ shows formation of smaller organoids and fewer connections made between them when SMAD4 has been re-expressed (+Dox) compared to organoids that have not had

SMAD4 re-expressed (-Dox). Connections are indicated by arrows. 4x images of days 3, 5, and 7 post onset of Doxycycline treatment for the Dox-Induced organoids are shown. Insets are expanded images of the boxed area in the original image. All three insets show the same organoids on their respective days. The scale bars shown are equivalent to 0.5 millimeters. Images are representative of four technical replicates and three biological replicates.

(B) Quantitative counts of connections made between organoids were normalized to the number of viable organoids in the well. The line shown is tumor organoid line SMAD4KO; BRAFV600E/+ induced with Doxycycline (+Dox) and uninduced (-Dox). Counts were done for days 3,5 and 7. Counts are representative of four technical replicates and three biological replicates. * = p<0.05, 2-Way ANOVA.

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Fig 3.(A) qPCR data of induced and uninduced SMAD4KO; BRAFV600E/+ tumor organoids is shown. Analysis was done for SMAD4 genes and its downstream targets. The induced organoids are denoted by ‘+Dox’ and uninduced

organoids are ‘-Dox’. Y-axis is (fold change/ -Dox organoids). The uninduced (-Dox) condition was normalized to 1. Values are representative of six technical replicates and three biological replicates. * = p<0.05, 2-Way ANOVA.

(B) qPCR data of induced and uninduced SMAD4KO; BRAFV600E/+ tumor organoids is shown. Analysis was done for genes associated with invasiveness in colon cancers such as genes with roles in the WNT signaling pathway, invadpodia formation, and extra-cellular matrix (ECM) degradation. The induced organoids are denoted by ‘+Dox’ and uninduced organoids are ‘-Dox’. Y-axis is (fold change/ -Dox organoids). The uninduced (-Dox) condition was normalized to 1. Values are representative of five technical replicates and three biological replicates.

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Fig 4. Diagram for visualization of target protein in SMAD4KO; BrafV600E/+ p-INDUCER tumor organoids. Organoids will be seeded (day 0) and cultured in CCM supplemented with either Vehicle (-Dox) or Doxycycline (+Dox). Organoids will be treated for 3 days, or until invasive phenotype forms (around Day 3). Organoids will be fixed, paraffin embedded, sectioned and probed for target protein and visualized under a microscope.

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