PROJECT PROGRESS REPORT for Mizutani Foundation Research Grant
Reference Number: 160178
Principal Investigator: Tongzhong JuOrganization: Emory University School of Medicine, Atlanta, GA 30322, USA
Period: April 1, 2016—June 30, 2017
Grant Title: The Role of Tn and STn Antigens in Progression and Metastasis of Colon Cancer(a) Abstract: Colorectal cancer (CRC) is a leading cause of cancer deaths in the Western world. To
understand its initiation, progression, and metastasis, many different animal models for CRC have been
established. Among them, mice with a germ line mutation in the gene encoding Adenomatous Polyposis Coli(APC) (APCmin/+) which were originally obtained in an ENU mutagenesis screen, is widely accepted in thefield because the gene with most predominant mutation in human CRC is APC. APCmin/+mice can develop
multiple intestinal neoplasia (min), yet without distal organ metastasis. Importantly, emerging evidenceshows that tumor associated carbohydrate antigens, Tn and sialylTn (STn) appear at an early stage of colon
carcinogenesis, and are associated with progression and metastasis. However, the molecular mechanismsunderlying the role of Tn/STn antigens in CRC tumorigenesis and progression are unknown. We recently
identified a novel X-linked gene Cosmc (C1GalT1C1 on human Xq24 and mouse Xc3), which encodes amolecular chaperone Cosmc regulating mucin O-glycosylation. Cosmc is required for folding of active core
1 b3-galactosyltransferase (T-synthase, C1GalT1 on 7p21.3), the key enzyme that converts Tn antigen tonormal core 1 O-glycans on glycoproteins in all animal cells. Many studies from ours and others showed thatCosmc mutations lead to loss of T-synthase activity and resultant expression of Tn/STn antigens in humancarcinoma cells and in other diseases. Remarkably, in our ongoing study, intestinal epithelial cell (IEC)specific Cosmc-knockout (IEC-Cosmc-KO) mice spontaneously developed invasive CRC. The molecularmechanisms underlying the colorectal carcinogenesis in these Cosmc-KO mice are through complex
pathways and require a comprehensive study. Objective: To investigate whether Tn/STn antigens promote
CRC formation, progression and ultimate metastasis to distal organs in the IEC-Cosmcnull: APCmin/+ CRC
animal model. Methods: using gene targeted strategy to generate IEC-Cosmcnull in APCmin/+ mice (IECCosmcnull:APCmi/+) and pathologically examine their primary CRC and metastatic tumors. Results: IECCosmcnull:APCmni/+ mice could not be generated due to the short life span of APCmin/+ mice. We had
successfully taken an alternative approach to generate IEC-APC:Cosmc-DKO mouse line by using APCtm1/+line. In the project period, 22 IEC-APC:Cosmc DKO mice (13 male and 9 female) and similar numbers ofcontrol mouse groups (APC-KO, Cosmc-KO and WT) in total were generated. Among them, the DKO maleand female mice grew and developed normally as other controls in their first 2 months, then slower growthwith smaller body size, stress signs, and mortality gradually occurred around 3-month age, while othercontrols IEC-APC-KO, IEC-Cosmc-KO and WT appeared normal. Four male DKO mice (IECAPCtm1/+:CosmcF/y:Cre+/-) (30%) died and a majority (8 of 9) of living male DKO mice at 3~4 moths age, anda fraction of (2 of 9) female DKO mice (IEC-APCtm1/+:CosmcF/+:Cre+/-) had to be sacrificed due to theirillness. DKO mice had significant lower survival rate (*P<0.01) compared to all other control groups.
Importantly, multiple gross tumors in the small intestine were observed in both APC-KO and APC:CosmcDKO mice, but the colorectal tumors were seen in all DKO mice and only in 15% of APC-KO mice.
Consistent with our early findings, thickened rectum was observed in Cosmc-KO male mice, but no tumor
was found in Cosmc+/- female mice. Furthermore, the enlarged inguinal lymph nodes were only found in theDKO mice. These results clearly indicate that the Cosmc deletion promotes the colorectal tumorigenesis in
APC-mutant mice. Unexpectedly, DKO mice suffered from severe anemia, likely due to an impaired ironhomeostasis because of dysfunctional iron-pump ferroportin on duodenum epithelia which could be possibly
O-glycosylated and degraded in DKO mice. Anemia of DKO mice might partially contribute to their slower
growth phenotype. HE staining of GI tract tissue sections confirmed the adenomas and adenocarcinomas in
the DKOs, and adenomas in APC-KOs and Cosmc-KO male mice. The significant progression andmetastasis of CRC were not observed thus far from these young mice, since it may require a longer time todevelop. Nevertheless, these preliminary results suggest that the expression of Tn/STn antigens significantly
promoted the colorectal carcinogenesis and perhaps progression in APC-mutant mice. More detailed

molecular analyses are underway, and the project will continue investigating the role of Tn/STn in CRC
progression and metastasis.
(b) Objectives: 1. To generate APC:Cosmc-double knockout (DKO), APC-KO (A-KO), and Cosmc-KO (CKO) mice;
2. To monitor and examine the development of colorectal tumors in the DKO, A-KO and C-KO animals;
3. To assess the malignancy of the colorectal tumors and possible metastatic tumors in those mice bypathology and molecular.
(c) Methods used: 1. Generating experimental animals using targeted gene-deletion technology: thebreeding strategy is shown below:

2. Assessing colorectal tumor development;
3. Analyzing the tumors including metastatic tumors by a pathological approach;
4. Analyzing Tn/STn Expression and tumor pathways by IHC: immunohistochemistry analysis on theTn/STn antigen expression, Wnt/APC/b-Catenin pathway, TGFb, and other pathways using specificantibodies against important players in these pathways.
(d) Results: At beginning of the project, the APCmin/+mice were used as an APC-mutant model as proposedin our original proposal. Yet, after ~4 months’ breeding, the double mutants, IEC-Cosmcnull:APCmin/+ mice
could not be generated due to the short life span (~3 months) and low productivity of APCmin/+ mouse line.
An alternative strategy (Figure) was immediately taken by replacing APCmin/+mouse line with APC-floxed(targeted mutation-1, APCtm1) mice, and worked well. In this period of research time, 22 of APC:CosmcDKO in total: 13 APCtm1/+:Cosmc F/y:Cre+/- (male) and 9 APCtm1/+:Cosmc F/+:Cre+/- (female), and similar
number of control male and female (APC-KO, Cosmc-KO and WT) animals were generated; they grewnormally at young ages (<2-months). Here are the major observations:
1. Gross Phenotypes: The male DKO mice started to show phenotypes when they grew older than 2-months:
slower growth or even body weight loss, curled back, and unthrilled; 4 of them died at ~3 months age; while
APC-KO, Cosmc-KO and WT controls developed normally. Eight of nine DKO male mice at age of 3~4
moths were sacrificed because of their illness, while all other control animals appeared to exhibit normal
growth with the exception of one Cosmc-KO mouse which developed rectal prolapse and had to beterminated. The female DKO mice also had similar but minor phenotypes than the male DKO mice; 6 of 9

female DKO lost weight after 3-months age, 3 of them had to be sacrificed at 4-months’ age due to illness,and DKO mice had a significantly lower survival rate (*P<0.01);
2. Tumors: multiple gross tumors grew in intestines in all DKO mice: 4~37 tumors with 0.1-0.3mm in sizein small intestines and 2~6 tumors with 0.1~0.5 mm in size in colorectum; enlarged brachial andinguinal lymph nodes were found in 6 of those 18 DKOs, but there was no obvious tumor mass in distantorgans such as liver and lung although they appeared pale. For 16 of APC-KO mice, 4~27 tumors wereobserved in their small intestines, but only 3 of them (16 total) had 1~2 gross tumors with sizes of 0.1-0.2
mm in colorectum. The thickened rectum was also observed in 9 of 10 male Cosmc-KO male mice;
3. Histology: H&E staining confirmed multiple adenoma and some carcinomas developed in both small
intestines and colorectum from DKO mice, but only adenomas were observed in small intestines (all APCKOs), and in colon (3 of 16 APC-KOs); epithelial morphology changes also appeared in Cosmc-KO mice,
mainly in rectum, consistent with our previous study;
4. Anemia: unexpectedly, all DKO mice suffered from severe anemia based on analysis of packed blood cellvolume (PCV: 20-30% in DKO versus 50% in control), while APC-KO and Cosmc-KO mice had a 10~15%
decrease in PCV. Surprisingly, Feroportin (FPN), an important iron pump glycoprotein expressed on GI-tractepithelial cells (mainly in duodenum epithelial cells) appeared to be largely degraded in the DKO mice,suggesting that the dysfunction of FPN might result in an impaired iron homeostasis in the DKO animal.
For the female DKO mice, because of the mosaicism of the Cosmc-deletion due to random X-chromosome
inactivation, the phenotype was milder than the male DKO mice, and the DKO females survived longer.
(e) Discussion: While the Tn and STn antigens are highly expressed in human colorectal carcinoma, which ismostly associated with gene mutations in the tumor suppressor APC, the role of Tn/STn in the development,progression and metastasis in conjunction with APC-mutation has not been investigated. In our study, for thefirst time, the expression of Tn/STn antigens was systematically investigated in an animal model with APC
mutation, which is the most relevant and key question in the colorectal tumor glycobiology. Our preliminary
results showed that the young IEC-APC:Cosmc-DKO mice developed more and larger colorectal tumors
compared to either APC-KO or Cosmc-KO mice, strongly indicating that Tn/STn antigens did play asignificant promoting role in colorectal carcinogenesis. In addition, unexpectedly, the DKO mice were also
found to suffer from severe anemia possibly due to an impaired iron homeostasis since the iron-pumpferroportin on duodenum epithelia was significantly degraded, which might also contribute to the slower
growth phenotype. While further molecular analyses are underway, this preliminary data is exciting andpromising. Although the metastatic tumors were not observed in distal organs in these young mice (<4.5months), they will be investigated in elder animals in a continued project. The preliminary study supportedby the Mizutani Foundation has laid a foundation for this exciting project, which will ultimately answer theimportant question regarding the role of most common tumor antigens, Tn/STn in malignancy of colorectal
carcinoma. This comprehensive study will have an important impact not only on our understanding ofrelevant tumor glyco-biology, but also on development of novel therapeutics in metastatic colon cancer infuture. In addition, this work also leads to a new exciting project on the role of O-glycosylation in ironhomeostasis, which is totally novel.
(f) List of publications: There is no publication yet.
(g) Name (signed) & Date:

7-28-2017