Fumihiko Kakizaki

Suppression of colon cancer metastasis by Aes through inhibition of Notch signaling

Metastasis is responsible for most cancer deaths. Here, we show that Aes (or Grg5) gene functions as an endogenous metastasis suppressor. Expression of Aes was decreased in liver metastases compared with primary colon tumors in both mice and humans. Aes inhibited Notch signaling by converting active Rbpj transcription complexes into repression complexes on insoluble nuclear matrix. In tumor cells, Notch signaling was triggered by ligands on adjoining blood vessels, and stimulated transendothelial migration. Genetic depletion of Aes in Apc(Δ716) intestinal polyposis mice caused marked tumor invasion and intravasation that were suppressed by Notch signaling inhibition. These results suggest that inhibition of Notch signaling can be a promising strategy for prevention and treatment of colon cancer metastasis.

Promotion of Colorectal Cancer Invasion and Metastasis through Activation of NOTCH–DAB1–ABL–RHOGEF Protein TRIO

UNLABELLED We have recently identified a metastasis suppressor gene for colorectal cancer: AES/Aes, which encodes an endogenous inhibitor of NOTCH signaling. When Aes is knocked out in the adenomatous epithelium of intestinal polyposis mice, their tumors become malignant, showing marked submucosal invasion and intravasation. Here, we show that one of the genes induced by NOTCH signaling in colorectal cancer is DAB1/Dab1. Genetic depletion of DAB1 suppresses cancer invasion and metastasis in the NOTCH signaling-activated mice. DAB1 is phosphorylated by ABL tyrosine kinase, which activates ABL reciprocally. Consistently, inhibition of ABL suppresses cancer invasion in mice. Furthermore, we show that one of the targets of ABL is the RAC/RHOGEF protein TRIO, and that phosphorylation at its Tyr residue 2681 (pY2681) causes RHO activation in colorectal cancer cells. Its unphosphorylatable mutation TRIO Y2681F reduces RHOGEF activity and inhibits invasion of colorectal cancer cells. Importantly, TRIO pY2681 correlates with significantly poorer prognosis of patients with colorectal cancer after surgery. SIGNIFICANCE These results indicate that TRIO pY2681 is one of the downstream effectors of NOTCH signaling activation in colorectal cancer, and can be a prognostic marker, helping to determine the therapeutic modality of patients with colorectal cancer.

Suppression of Colonic Polyposis by Homeoprotein CDX2 through its Nontranscriptional Function that Stabilizes p27Kip1

Caudal-related homeoprotein CDX2 is expressed in intestinal epithelial cells, in which it is essential for their development and differentiation. A tumor suppressor function is suggested by evidence that CDX2 levels are decreased in human colon cancer specimens and that an inactivating mutation of Cdx2 in Apc(Δ716) mice markedly increases the incidence of colonic polyps. In this study, we investigated roles for transcriptional and nontranscriptional functions of CDX2 in suppression of colonic tumorigenesis. Mutagenic analysis of CDX2 revealed that loss of function stabilizes CDK inhibitor p27Kip1 by a nontranscriptional but homeodomain-dependent mechanism that inhibits cyclin E-CDK2 activity and blocks G0/G1-S progression in colon cancer cells. p27Kip1 stabilization was mediated by an inhibition of ubiquitylation-dependent proteolysis associated with decreased phosphorylation of Thr187 in p27Kip1. siRNA-mediated knockdown of p27Kip1 relieved the decrease in cyclin E-CDK2 activity and S-phase cell fraction elicited by CDX2 expression. Together, these results implicate a nontranscriptional function of CDX2 in tumor suppression mediated by p27Kip1 stabilization. Up to approximately 75% of low-CDX2 human colon cancer lesions show reduced levels of p27Kip1, whereas approximately 68% of high-CDX2 lesions retain expression of p27Kip1. These results show that low levels of CDX2 accelerate colon tumorigenesis by reducing p27Kip1 levels.

CDX Transcription Factors Positively Regulate Expression of Solute Carrier Family 5, Member 8 in the Colonic Epithelium

BACKGROUND & AIMS Caudal-related homeodomain transcription factors CDX1 and CDX2 regulate gut development and differentiation of intestinal epithelial cells; they are candidate tumor suppressors of colorectal carcinomas. Because the functions of CDX1 and CDX2 in the colonic epithelium are not fully understood, we sought to identify genes that they target. METHODS We conducted a chromatin immunoprecipitation (ChIP) screen to identify genes that bind the CDX transcription factors. Expression of target genes was analyzed in colon cells and tissues from Cdx1(-/-), Cdx2(+/-), Apc(+/Delta716), and wild-type (control) mice. RESULTS Using the ChIP screen, we identified solute carrier family 5, member 8 (SLC5A8, also known as SMCT1) as a direct target of CDX1 and CDX2. CDX transcription factors bind to the promoter region of SLC5A8 and transactivate SLC5A8 reporter constructs. Overexpression of Cdx1 or Cdx2 in human colon cancer cell lines induced expression of endogenous SLC5A8, whereas CDX1 and CDX2 knockdowns reduced its level. Consistently, Slc5a8 expression was significantly reduced in colons of Cdx1(-/-) or Cdx2(+/-) mice compared with wild-type mice. Slc5a8 levels were also reduced in colonic adenomatous polyps and hamartomas from Apc(+/Delta716) and Cdx2(+/-) mutant mice, respectively, compared with adjacent normal colon tissues. CONCLUSIONS CDX1 and CDX2 bind the promoter region of SLC5A8 and up-regulate its expression in cultured cells and in colonic epithelium. SLC5A8 transports monocarboxylates such as pyruvate, lactate, and butyrate; CDX1 and CDX2 might therefore regulate the uptake of these substances in the colon.

Amino-terminal enhancer of split gene AES encodes a tumor and metastasis suppressor of prostate cancer

A major cause of cancer death is its metastasis to the vital organs. Few effective therapies are available for metastatic castration‐resistant prostate cancer (PCa), and progressive metastatic lesions such as lymph nodes and bones cause mortality. We recently identified AES as a metastasis suppressor for colon cancer. Here, we have studied the roles of AES in PCa progression. We analyzed the relationship between AES expression and PCa stages of progression by immunohistochemistry of human needle biopsy samples. We then performed overexpression and knockdown of AES in human PCa cell lines LNCaP, DU145 and PC3, and determined the effects on proliferation, invasion and metastasis in culture and in a xenograft model. We also compared the PCa phenotypes of Aes/Pten compound knockout mice with those of Pten simple knockout mice. Expression levels of AES were inversely correlated with clinical stages of human PCa. Exogenous expression of AES suppressed the growth of LNCaP cells, whereas the AES knockdown promoted it. We also found that AES suppressed transcriptional activities of androgen receptor and Notch signaling. Notably, AES overexpression in AR‐defective DU145 and PC3 cells reduced invasion and metastasis to lymph nodes and bones without affecting proliferation in culture. Consistently, prostate epithelium‐specific inactivation of Aes in Ptenflox/flox mice increased expression of Snail and MMP9, and accelerated growth, invasion and lymph node metastasis of the mouse prostate tumor. These results suggest that AES plays an important role in controlling tumor growth and metastasis of PCa by regulating both AR and Notch signaling pathways.

An improved method for culturing patient-derived colorectal cancer spheroids

Recent advances allowed culturing and examination of patient-derived colorectal cancer (PD-CRC) cells as organoids or spheroids. To be applied to practical personalized medicine, however, current methods still need to be strengthened for higher efficiency. Here we report an improved method to propagate PD-CRC tumor initiating cells (TICs) in spheroid culture. We established > 100 cancer spheroid lines derived from independent colorectal cancer patients employing a serum-containing medium with additional inhibitors, Y27632 and SB431542. Because colorectal cancer spheroids showed wide-range growth rates depending on the patient tumors, we searched for supplementary factors that accelerated proliferation of slow-growing CRC-TIC spheroids. To this end, we introduced a convenient growth-monitoring method using a luciferase reporter. We found that epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) were critical for steady propagation of a subset of CRC-TIC spheroids carrying the wild-type RAS and RAF genes. We also identified 5′-(N-ethyl-carboxamido)-adenosine (NECA), an adenosine receptor agonist, as an essential supplement for another subset of spheroids. Based on these results, we propose to optimize culture conditions for CRC-TIC spheroids by adjusting to the respective tumor samples. Our method provides a versatile tool that can be applied to personalized chemotherapy evaluation in prospective clinical studies.

Characterization of Aes nuclear foci in colorectal cancer cells

Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling.

Id2 deletion attenuates Apc-deficient ileal tumor formation

ABSTRACT The expression level of inhibitor of DNA binding 2 (Id2) is increased in colorectal carcinomas and is positively correlated with poor prognosis. However, the functional significance of Id2 in intestinal tumorigenesis has not been fully defined using genetic approaches. Here, we show that Id2 promotes ileal tumor initiation in Apc-deficient mice. Expression of Id2 was stimulated by Wnt signaling through the enhancer region of the Id2 promoter at the early stage of tumorigenesis in Apc+/Δ716 (ApcΔ716) mice. Genetic depletion of Id2 in ApcΔ716 mice caused ∼80% reduction in the number of ileal polyps, but had little effect on tumor size. Notably, the lack of Id2 increased the number of apoptotic cells in the normal crypt epithelium of the mice. Furthermore, DNA microarray analysis revealed that the expression level of Max dimerization protein 1 (Mxd1), known as a c-Myc antagonist, was specifically increased by Id2 deletion in the ileal intestinal epithelium of ApcΔ716 mice. In contrast, the protein level of c-Myc, but not the mRNA level, was decreased by loss of Id2 in these mice. These results indicate that loss of Id2 inhibits tumor initiation by up-regulation of Mxd1 and down-regulation of c-Myc in ApcΔ716 mice.

Expression of metastasis suppressor gene AES driven by a YY element in a CpG island promoter and transcription factor YY2.

We recently found that the product of the AES gene functions as a metastasis suppressor of colorectal cancer (CRC) in both humans and mice. Expression of amino‐terminal enhancer of split (AES) protein is significantly decreased in liver metastatic lesions compared with primary colon tumors. To investigate its downregulation mechanism in metastases, we searched for transcriptional regulators of AES in human CRC and found that its expression is reduced mainly by transcriptional dysregulation and, in some cases, by additional haploidization of its coding gene. The AES promoter‐enhancer is in a typical CpG island, and contains a Yin‐Yang transcription factor recognition sequence (YY element). In human epithelial cells of normal colon and primary tumors, transcription factor YY2, a member of the YY family, binds directly to the YY element, and stimulates expression of AES. In a transplantation mouse model of liver metastases, however, expression of Yy2 (and therefore of Aes) is downregulated. In human CRC metastases to the liver, the levels of AES protein are correlated with those of YY2. In addition, we noticed copy‐number reduction for the AES coding gene in chromosome 19p13.3 in 12% (5/42) of human CRC cell lines. We excluded other mechanisms such as point or indel mutations in the coding or regulatory regions of the AES gene, CpG methylation in the AES promoter enhancer, expression of microRNAs, and chromatin histone modifications. These results indicate that Aes may belong to a novel family of metastasis suppressors with a CpG‐island promoter enhancer, and it is regulated transcriptionally.

Abstract B65: Promotion of colorectal cancer invasion and metastasis through activation of Notch–Dab1–Abl–RhoGEF protein trio

We have recently identified a metastasis suppressor gene for colorectal cancer: AES/Aes that encodes an endogenous inhibitor of Notch signaling (1). When Aes is knocked out in the adenomatous epithelium of intestinal polyposis mice, their tumors become malignant, showing marked submucosal invasion and intravasation. Here, we show that one of the genes induced by NOTCH signaling in colorectal cancer is DAB1/Dab1. Genetic depletion of DAB1 suppresses cancer invasion and metastasis in the NOTCH signaling-activated mice. DAB1 is phosphorylated by ABL tyrosine kinase, which activates ABL reciprocally. Consistently, inhibition of ABL suppresses cancer invasion in mice. Furthermore, we show that one of the targets of ABL is the RAC/RHOGEF protein TRIO, and that phosphorylation at its Tyr residue 2681 (pY2681) causes RHO activation in colorectal cancer cells. Its unphosphorylatable mutation TRIO Y2681F reduces Rho-GEF activity and inhibits invasion of colorectal cancer cells. Importantly, Trio (pY2681) correlates with significantly poorer prognosis of patients with colorectal cancer after surgery (2). These results indicate that Trio(pY2681) is one of the downstream effectors of Notch signaling activation in colorectal cancer, and can be a prognostic marker, helping to determine the therapeutic modality of patients with colorectal cancer. More recent results will be discussed. (1) Cancer Cell; 19: 125–137, 2011. (2) Cancer Discov; 5:198–211, 2015. Citation Format: Masahiro Sonoshita, Yoshiro Itatani, Fumihiko Kakizaki, Yoshiharu Sakai, M. Mark Taketo. Promotion of colorectal cancer invasion and metastasis through activation of Notch–Dab1–Abl–RhoGEF protein trio. [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 B65.