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MRTFB Suppresses Colorectal Cancer Development Through Regulating SPDL1 and MCAM

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MRTFB Suppresses Colorectal Cancer Development Through Regulating SPDL1 and MCAM MRTFB suppresses colorectal cancer development through regulating SPDL1 and MCAM Takahiro Kodamaa,b,c, Teresa A. Mariana,b, Hubert Leea, Michiko Kodamaa, Jian Lid, Michael S. Parmacekd, Nancy A. Jenkinsa,e, Neal G. Copelanda,e,1, and Zhubo Weia,b,1 aHouston Methodist Research Institute, Houston Methodist Hospital, Houston, TX 77030; bHouston Methodist Cancer Center, Houston Methodist Hospital, Houston, TX 77030; cDepartment of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, 5650871 Suita, Osaka, Japan; dDepartment of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104; and eGenetics Department, The University of Texas MD Anderson Cancer Center, Houston, TX 77030 Contributed by Neal G. Copeland, October 7, 2019 (sent for review June 18, 2019; reviewed by Masaki Mori and Hiroshi Seno) Myocardin-related transcription factor B (MRTFB) is a candidate tumor- shown to regulate cell cycle progression (9) and HCC xenograft suppressor gene identified in transposon mutagenesis screens of the tumor growth (10). Functional validation using cell culture sys- intestine, liver, and pancreas. Using a combination of cell-based assays, tems have also shown that reduced expression of MRTFB by in vivo tumor xenograft assays, and Mrtfb knockout mice, we demon- RNA interference leads to increased CRC cell invasion (4), strate here that MRTFB is a human and mouse colorectal cancer suggesting its important role in tumor progression. (CRC) tumor suppressor that functions in part by inhibiting cell Based on these results, we decided to conditionally delete invasion and migration. To identify possible MRTFB transcriptional Mrtfb in the mouse intestine to further explore its role in CRC. targets, we performed whole transcriptome RNA sequencing in We found that tumor growth was significantly accelerated in the MRTFB siRNA knockdown primary human colon cells and identi- mouse intestine on Mrtfb knockout, which is critical for func- fied 15 differentially expressed genes. Among the top candidate tional validation. We also showed that knockdown of MRTFB tumor-suppressor targets were melanoma cell adhesion molecule expression in human CRC cells resulted in accelerated xenograft (MCAM), a known tumor suppressor, and spindle apparatus coiled-coil tumor growth as well as increased invasion and migration of protein 1 (SPDL1), which has no confirmed role in cancer. To determine human CRC cells. We then performed whole transcriptome whether these genes play a role in CRC, we knocked down the expres- RNA sequencing using primary human colon cells with reduced sion of MCAM and SPDL1 in human CRC cells and showed significantly MRTFB expression to identify MRTFB downstream genes, GENETICS increased invasion and migration of tumor cells. We also showed that which led to the identification of a number of genes, including a Spdl1 expression is significantly down-regulated in Mrtfb knockout known tumor suppressor, MCAM (melanoma adhesion mole- mouse intestine, while lower SPDL1 expression levels are significantly cule), and a candidate CRC tumor suppressor, SPDL1 (spindle associated with reduced survival in CRC patients. Finally, we show that apparatus coiled-coil protein 1). Subsequent follow-up studies depletion of MCAM and SPDL1 in human CRC cells significantly in- showed that SPDL1 has tumor-suppressor activities in both cell- creases tumor development in xenograft assays, further confirming based and xenograft assays using human CRC cells, and that their tumor-suppressive roles in CRC. Collectively, our findings demon- reduced SPDL1 expression levels are significantly associated strate the tumor-suppressive role of MRTFB in CRC and identify several with shorter overall survival in human CRC patients. Collec- genes, including 2 tumor suppressors, that act downstream of MRTFB to tively, our studies have identified the human CRC tumor sup- regulate tumor growth and survival in CRC patients. pressor MRTFB, as well as several MRTFB downstream genes, including MCAM and SPDL1. MRTFB | colorectal cancer | tumor suppressor | RNA-seq | SPDL1 Significance olorectal cancer (CRC) is the second-leading cause of Ccancer-related deaths worldwide, with 860,000 deaths and Myocardin-related transcription factor B (MRTFB) is a candidate 1.8 million newly diagnosed cases each year (1). CRC imposes a tumor-suppressor gene identified in transposon mutagenesis large burden on the health care system, with approximately $14 screens of the intestine, liver, and pancreas. Using a combina- billion spent annually to treat CRC in the US alone (2). Therefore, tion of cell-based assays, in vivo tumor xenograft assays, and numerous studies have attempted to understand the etiology of Mrtfb knockout mice, we demonstrate that MRTFB is a mouse CRC and apply the research findings to patient care and treatments. and human colorectal cancer (CRC) tumor-suppressor gene that In the last several years, cancer genomics has become a functions in part by inhibiting cell invasion and migration. Us- promising tool for furthering our understanding of CRC. Rep- ing whole transcriptome RNA sequencing in Mrtfb-knockdown resentative studies include The Cancer Genome Atlas molecular cells, we also identify several MRTFB downstream genes, in- characterization of hundreds of CRCs to identify significantly cluding a known tumor suppressor, MCAM, and a candidate mutated genes (3) and Sleeping Beauty (SB) transposon muta- tumor suppressor, SPDL1. Finally, we show that MCAM and genesis screens designed to uncover new CRC candidate driver SPDL1 are also human CRC tumor-suppressor genes that act genes in mouse models of CRC (4). A large number of candidate downstream of MRTFB to regulate CRC growth and survival. cancer driver genes have been identified by these studies, many Author contributions: Z.W. designed research; T.K., T.A.M., H.L., M.K., and Z.W. per- of which have unknown roles in CRC, such as myocardin- related formed research; J.L. and M.S.P. contributed new reagents/analytic tools; N.A.J., N.G.C., transcription factor B (MRTFB). Mrtfb also has been identified and Z.W. analyzed data; and N.G.C. and Z.W. wrote the paper. as a candidate cancer driver gene in other transposon mutagenesis Reviewers: M.M., Kyushu University; and H.S., Kyoto University. screens in the gastrointestinal (GI) tract, including hepatocellular The authors declare no competing interests. carcinoma (HCC) (5) and pancreatic ductal adenocarcinoma Published under the PNAS license. (PDAC) (6). These studies suggest a potentially important role for 1To whom correspondence may be addressed. Email: [email protected] or Mrtfb in GI tract cancers. [email protected]. Mrtfb is an essential gene, as its whole-body knockout in mice This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. leads to embryonic lethality at around embryonic day (E) 13.5, 1073/pnas.1910413116/-/DCSupplemental. due mainly to cardiovascular defects (7, 8). Mrtfb has also been First published November 5, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1910413116 PNAS | November 19, 2019 | vol. 116 | no. 47 | 23625–23635 Downloaded by guest on September 29, 2021 Results Knockout of Mrtfb in Mouse Intestine Significantly Enhances Tumor Mrtfb Is a Candidate Driver Gene for GI Tract Cancers. SB transposon Development in Apc Mutant Mice. Since tumor development in mutagenesis screens performed in the intestines of mice carrying knockout mice is critical for functional validation of a tumor- suppressor gene, we decided to knock out Mrtfb in the mouse sensitizing mutations in genes that act at different stages of CRC + + + Flox Cre/ development, including APC (Apcmin/ ), KRAS (KrasG12D/ ), intestine using a conditional Mrtfb allele (8). Lrig1 was KO/+ R172H/+ used as the Cre driver to specifically induce deletion of both SMAD4 (Smad4 ), and TP53 (p53 ), identified 11 + copies of Mrtfb in the intestine, while ApcFlox/ wasusedtosensi- candidate tumor-suppressor genes that were mutated in all 4 + tize these mice to intestinal tumor development (21). Lrig1Cre/ ; cohorts (4), suggesting their critical roles in CRC. Seven of these + + + + + ApcFlox/ ;MrtfbFlox/Flox and Lrig1Cre/ ;ApcFlox/ ;Mrtfb / mice are established cancer driver genes, including Abl1 (11), were generated and treated with tamoxifen by intraperitoneal Ankrd11 (12), Arid1a (13), Ctnna1 (14), Gnb1 (15), Pik3r1 (16), injection (IP) at 2 mg/d for 3 consecutive days, beginning at age and Zfp148 (17). Mrtfb was 1 of the 4 candidate tumor- 6 to 8 wk, to activate Cre expression. Knockout of Mrtfb ex- + + suppressor genes identified, along with Dennd4c, Luc7l2, and pression in Lrig1Cre/ ;ApcFlox/ ;MrtfbFlox/Flox intestine was sub- Ppm1b, that have no clarified role in CRC or other types of sequently confirmed by quantitative reverse transcription cancer. However, a potentially important role for Mrtfb in GI polymerase chain reaction (RT-qPCR) (Fig. 3A), and the mice tract cancer has been further suggested by other SB transposon were monitored for tumor development at 4, 5, and 6 mo after mutagenesis screens, which identified Mrtfb as a candidate tamoxifen injection. As shown in Fig. 3B, control animals de- tumor-suppressor gene in HCC (5) and PDAC (6). Therefore, veloped an average of 2.4 tumors at the 4-mo time point, while we focused on Mrtfb in the present study. Mrtfb knockout mice developed 7.8 tumors, a 3-fold increase. At The insertion pattern of SB transposons in a given gene is the 5-mo time point, we observed an average of ∼13 tumors in indicative of whether it is functioning as an oncogene or a tumor- each Mrtfb knockout mouse, compared with only ∼4 tumors in suppressor gene (18–20). As shown in Fig. 1A, transposon in- control animals (Fig. 3 C and D). A similar acceleration of tumor sertions in Mrtfb in mouse intestinal tumors are distributed al- development was observed at the 6-mo time point (Fig. 3E), fur- most evenly across the gene and in both orientations, which is ther confirming Mrtfb’s role as a CRC tumor suppressor. indicative of a tumor-suppressor gene. Consistent with this, We also collected tumor samples from control and Mrtfb lower MRTFB expression levels are associated with shorter knockout mice.
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