Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and colorectal cancers

    Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and colorectal cancers Ju Hwa Lee BS, Youn Jin Choi MD, Eun Mi Je MS, Ho Shik Kim MD, Nam Jin Yoo MD, Sug Hyung Lee MD PII: DOI: Reference:

S0046-8177(15)00502-X doi: 10.1016/j.humpath.2015.12.009 YHUPA 3773

To appear in:

Human Pathology

Received date: Revised date: Accepted date:

28 October 2015 2 December 2015 11 December 2015

Please cite this article as: Lee Ju Hwa, Choi Youn Jin, Je Eun Mi, Kim Ho Shik, Yoo Nam Jin, Lee Sug Hyung, Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and colorectal cancers, Human Pathology (2015), doi: 10.1016/j.humpath.2015.12.009

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ACCEPTED MANUSCRIPT Revision YHUPA-D-15-00676 Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and

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colorectal cancers

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Ju Hwa Lee BSa, Youn Jin Choi MDa, Eun Mi Je MSa, Ho Shik Kim MDb, Nam Jin Yoo MDa, * and Sug Hyung Lee MDa, *

of Korea, Seoul 137-701, Korea

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Departments of aPathology and bBiochemistry, College of Medicine, The Catholic University

Running title: WISP3 mutation in cancer

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Key words: WISP3, colon cancer, somatic mutation, gastric cancer, heterogeneity

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* Address correspondence to: Sug Hyung Lee, MD, Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701,

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Or

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Korea. E-mail: [email protected]

Nam Jin Yoo, MD, Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea. E-mail: [email protected]

Conflict of interest statement: All of the authors declare the absence of any conflict of interest.

ACCEPTED MANUSCRIPT Abstract WISP3 is involved in many cancer-related processes including epithelial-mesenchymal

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transition, cell death, invasion and metastasis and is considered a tumor suppressor. The aim

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of our study was to find whether WISP3 gene was mutated and expressionally altered in gastric (GC) and colorectal cancers (CRCs). WISP3 gene possesses a mononucleotide repeat

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in the coding sequence that could be mutated in cancers with high microsatellite instability

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(MSI-H). We analyzed 79 GCs and 156 CRCs, and found that GCs (8.8%) and CRCs (10.5%) with MSI-H, but not those with microsatellite stable/low MSI (MSS/MSI-L), harbored a frameshift mutation. We also analyzed intratumoral heterogeneity (ITH) of the frameshift mutation in 16 CRCs and found that the WISP3 mutation exhibited regional ITH in 25% of

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the CRCs. In immunohistochemistry, loss of WISP3 expression was identified in 24% of GCs and 21% of CRCs. The loss of expression was more common in those with WISP3 mutation

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than with wild-type WISP3 and those with MSI-H than with MSS/MSI-L. Our data indicate

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WISP3 harbored not only frameshift mutation but also mutational ITH and loss of expression, which together might play a role in tumorigenesis of GC and CRC with MSI-H by inhibiting

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tumor suppressor functions of WISP3. Our data also suggest that mutation analysis in multiregions is needed for a proper evaluation of mutation status in GC and CRC with MSI-H.

Key words: WISP3, colon cancer, somatic mutation, gastric cancer, heterogeneity

ACCEPTED MANUSCRIPT Introduction It is now well believed that epithelial-mesenchymal transition (EMT) is actively involved in

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both cancer development and progression [1, 2]. During EMT, epithelial cells lose cell-cell

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adhesion, and gain migratory and invasive properties to become mesenchymal cells [1-3]. Alteration of cytoskeletal proteins, especially loss of E-cadherin, is considered to be an

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essential event in EMT. Transcription factors, including SNAI1, SNAI2, ZEB1 and ZEB2,

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repress E-cadherin expression and subsequently activate EMT [4-8]. ZEB1 is induced by multiple signaling pathways including TGF- β, Notch and Wnt. WNT1-inducible-signaling pathway protein 3 (WISP3) is a member of the WISP protein subfamily, which mediate diverse developmental processes. WISP3 attenuates IGF-1 to

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decrease ZEB1 expression and EMT, subsequently inhibiting invasion of breast cancers [9]. Experimentally, blockade of WISP3 activates cell survival and enhances resistance to cell

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death in mammary epithelial cells [10]. These data suggest that WISP3 might be a tumor

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suppressor gene (TSG). An earlier study identified frameshift mutations of WISP3 gene within the coding mononucleotide repeat (A9) in colorectal cancers (CRCs) [11] and a rare

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type of breast cancer [12].

Mononucleotide repeats are frequently mutated in cancers with microsatellite instability (MSI). Frameshift mutations in mononucleotide repeats are features of gastric cancer (GC), CRC and endometrial cancer with microsatellite instability (MSI) [13, 14]. Possibly, frameshift mutations in WISP3 might cause inactivation of its TSG functions and contribute to cancer pathogenesis. Although the mutation within the A9 repeat of WISP3 has been reported in CRCs [11], it has not been reported in GCs. A cancer usually acquires intratumoral heterogeneity (ITH) after clonal expansion, which may impede proper diagnosis and therapy [15, 16]. Mutational ITH of the WISP3 frameshift mutation has not been studied in cancers. In this study, we analyzed WISP3 frameshift mutation, mutational ITH and

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expression in gastrointestinal cancers with MSI-H (GCs and CRCs).

ACCEPTED MANUSCRIPT Materials and method Tissue samples and microdissection

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For the mutation analysis, sporadic 79 GCs and 156 CRCs from Koreans were used in this

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study. Of them, 55 CRCs were frozen tissues and the other 180 tissues were methacarn-fixed tissues. The GCs consisted of 34 GCs with high MSI (MSI-H), 45 GCs with microsatellite

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stable/low MSI (MSS/MSI-L), 76 CRCs with MSI-H and 80 CRCs with MSS/MSI-L. The

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MSI evaluation system used five mononucleotide repeats (BAT25, BAT26, NR-21, NR-24 and MONO-27), tumoral MSI status of which was characterized as: MSI-H, if two or more of these markers show instability, MSI-L, if one of the markers shows instability and MSS, if none of the markers shows instability [17]. For 55 CRCs, we collected four to seven different

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tumor areas and one normal mucosal area from each fresh CRC specimen to analyze the mutational ITH. The tumor areas were 0.027-1 cm3 and at least 1.0 cm apart from each other.

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To confirm that these multi-regional biopsies were all areas of carcinoma (as opposed to areas

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of normal or dysplasia), they were frozen, stained with hematoxylin and eosin and examined under light microscope. The tumor cell purities of the ITH tissues were at least 70%. Sixteen

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of the 55 CRCs with ITH collection were identified as MSI-H. Pathologic features of the cancers are summarized in Table 1. The histologic features of CRC with MSI-H, including mucinous histology, tumor infiltrating lymphocytes, medullary pattern, and Crohn's like inflammation, were evaluated in all blocks of all cases by a pathologist. Malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection as described previously [18, 19]. DNA extraction was performed by a modified single-step DNA extraction method by proteinase K treatment. Approval of this study was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study.

ACCEPTED MANUSCRIPT Single strand conformation polymorphism (SSCP) analysis WISP3 has a mononucleotide repeat (A9 repeat) in their coding sequences (exon 5). Genomic

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DNA from the microdissected cells was isolated, and was amplified by polymerase chain

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reaction (PCR) with specific primer pairs. Radioisotope ([32P]dCTP) was incorporated into the PCR products for detection by autoradiogram. After SSCP, mobility shifts on the SSCP

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gels (FMC Mutation Detection Enhancement system; Intermountain Scientific, Kaysville, UT,

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USA) were determined by visual inspection. Direct DNA sequencing reactions in both forward and reverse sequences were performed in the cancers with the mobility shifts in the SSCP using a capillary automatic sequencer (3730 DNA Analyzer, Applied Biosystem, Carlsbad, CA, USA). When mutations in the genes were suspected by SSCP, analysis of an

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independently isolated DNA from another tissue cut of the same patients was performed to exclude potential artifacts originated from PCR. Other procedures for PCR-SSCP were

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described in our previous reports [18, 19].

Immunohistochemistry

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Using the sections from GC and CRC tissues, immunohistochemistry for WISP3 was performed. The tissues consisted of 34 GCs and 76 CRCs with MSI-H, and 45 GCs and 80 CRCs with MSS/MSI-L. We used ImmPRESS System (Vector Laboratories, Burlingame, CA, USA) and antibody for human WISP3 (GeneTex, Irvine, CA, USA; dilution 1/50). The immunohistochemistry procedure was performed as described previously [20]. The reaction products were developed with diaminobenzidine and counterstained with hematoxylin. The staining intensity was graded as follows: 0, negative; 1+ when the cells showed weak staining in cytoplasm; 2+, moderate; and 3+, intense. The extent was graded according to the percentage of positive cells as follows: 0, 0–5%; 1, 6-19%; 2, 20–49%; 3, > 50%. The percentage of positive cells and the staining intensity were then multiplied to generate the

ACCEPTED MANUSCRIPT immunohistochemistry score (IS). We categorized the IS 0-2 as negative, 3 or 4 as + and 6 or 9 as ++. Both + and ++ were considered positive. The immunostaining was judged to be

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specific by absence of consistent immunostaining of cells with replacement of primary

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antibody with the blocking reagent and reduction of immunostaining intensity as dilution of the antibody was increased. For the statistical analysis of the immunohistochemical data, we

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used Fisher’s exact test and χ2 test.

ACCEPTED MANUSCRIPT Results Mutational analysis

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Genomic DNAs isolated from normal and tumor tissues of the 79 GCs and 156 CRCs were

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analyzed for detection of mutation in WISP3 (A9 repeat) by PCR-SSCP analysis. On the SSCP, we observed aberrant bands in 11 cases of the cancers (Figure 1 and Table 2). PCR

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products from matched normal tissues showed no evidence of aberrant migration in the SSCP,

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indicating the aberrant migration had risen somatically (Figure 1). Direct Sanger sequencing of the cancer DNA confirmed that they represented somatic mutations of WISP3 gene (Figure 1). All of the mutations were considered heterozygous mutations according to SSCP and sequencing analyses (Figure 1). They were an identical deletion mutation of one base within

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the A9 repeat that would cause a premature stop codon (Table 2). Three of 34 GCs (8.8%) and eight of 76 CRCs (10.5%) with MSI-H harbored the WISP3 frameshift mutation. In the

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mutated cases, Sanger sequencing analyses were performed using DNA from another tissue

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cuts and they showed the same mutation. WISP3 frameshift mutations were found in MSI-H GCs and CRCs, but not in those with

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MSS/MSI-L (Table 2). There was a statistical difference in the WISP3 frameshift mutation frequencies between the cancers with MSI-H (11/110) and MSS/MSI-L (0/125) (Fisher’s exact test, p < 0.001). There was no significant difference in the WISP3 mutation between GCs and CRCs (Fisher’s exact test, p > 0.05). There was no significant association of WISP3 frameshift mutations with pathologic data of the patients (age, sex, histologic grade and stage) (χ2 test, p > 0.05).

Intratumoral heterogeneity of WISP3 frameshift mutation Ninety-four fragments from 16 CRCs with MSI-H (4-7 fragments per case) were analyzed with respect to regional status of the WISP3 frameshift mutation. They were detected in four

ACCEPTED MANUSCRIPT CRCs (25.0%) (Table 3). Interestingly, all of the four exhibited ITH of the WISP3 mutation (#43, 45, 48 and 49) (Figure 2). In each of the four CRCs, the mutation was detected in one of

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the regions (Figure 2). We could not find any histological difference among the ITH regions.

Immunohistochemical analysis

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To identify whether WISP3 was altered at protein level, we studied WISP3 expression in 34

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GCs and 76 CRCs with MSI-H, and 45 GCs and 80 CRCs with MSS/MSI-L by immunohistochemistry (Figure 3). WISP3 immunostainings, when present, were observed in cytoplasm (Figure 3). Negative controls showed no immunostainings in the tissues. WISP3 was positively expressed (IS 6 or 9) in both non-neoplastic gastric and colonic mucosal cells

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(Figure 3A and 3D). In the cancers, immunopositivity for WISP3 was observed in 60 (75.9%) of the 79 GCs and 123 (78.8%) of the 156 CRCs (Table 4, Figure 3B and 3E). Positive

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WISP3 expression in GCs with MSI-H (22/34) was significantly lower than that in those with

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MSI-L/MSS (38/45) (Fisher’s exact test, p = 0.039). Positive WISP3 expression in CRCs with MSI-H (51/76) was significantly lower than that in those with MSI-L/MSS (72/80) (Fisher’s

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exact test, p < 0.001).

Seven of eight CRCs and all three GCs with WISP3 frameshift mutation displayed negative WISP3 immunostaining (IS 0-3) (Figure 3C and F). Positive WISP3 immunostaining in MSI-H cancers with WISP3 frameshift mutation (1/11) was significantly lower than that without WISP3 frameshift mutations (182/224) (Fisher’s exact test, p < 0.001). MSI-H GCs and CRCs with WISP3 frameshift mutation exhibited significantly lower WISP3 immunopositivity than MSI-H GCs and CRCs devoid of WISP3 frameshift mutation, respectively (Table 4). The WISP3 expression was not significantly different with respect to the origin (GC vs. CRC) (Fisher’s exact test, p > 0.05). On initial evaluation, two pathologists had disagreement in some cases (Cohen’s kappa coefficient; 0.77), but they examined the

ACCEPTED MANUSCRIPT slides together and finally reached to an agreement. There was no association of WISP3 expression with age, sex, depth of invasion and stages (χ2 test, p > 0.05).

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Discussion

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In this study, we found a significant difference in mutational and expressional prevalence of WISP3 between MSI-H and MSS/MSI-L cancers. Also, we identified ITH of the WISP3

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frameshift mutation in MSI-H CRCs. Together, these data suggest that WISP3 gene

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perturbation by frameshift mutation and expression loss might be a feature of MSI-H GC and CRC. WISP3 frameshift mutation was a deletion mutation that resembled a loss-of-function mutation. Based on the earlier knowledges of WISP3 (inhibition of EMT, cell death, invasion and metastasis of cancer cells) [9, 10, 21], the data suggest that WISP3 inactivation may

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possibly activate these processes and possibly contribute to oncogenic activity. WISP3 immunostaining was negative in the cancers harboring the frameshift mutation

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except one case (Table 4). As anti-WISP3 antibody used in the present study had been

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produced using a synthetic peptide of amino acids 320-335 of human WISP3. This sequence area would be cut off by the WISP3 mutation (p.Lys208AsnfsX24) and the mutated WISP3

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could not be detected by the antibody. Sanger sequencing data indicate that all of the WISP3 mutations appeared heterozygous ones (Figure 1), strongly suggesting that the second alleles without mutations were intact. The WISP3 loss in WISP3-mutated cases appeared to come from the mutation of one allele and other mechanisms of the second allele. Another option is that quantity of WISP3 from the second allele alone may not be enough for the detection. WISP3 mutations in our cohort (Asian) were identified in 8 cases (10.5%) of 76 MSI-H CRC cases. In contrast, the WISP3 mutation rate was 30.6% in MSI-H CRCs in the previous study (European) [11]. There is a statistical difference of the mutation frequencies between them (p = 0.01). These data suggest that there could be a racial difference in the WISP3 mutation or a case selection bias or a technical bias. For the mutation detection, we used

ACCEPTED MANUSCRIPT conventional denaturing gel electrophoresis (SSCP) and Sanger sequencing while the previous study adopted fluorescent-labeled PCR and capillary electrophoresis. To see whether

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the incidence of WISP3 mutation varies depending on ethnicity, studies are now needed that

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attempt to find WISP3 mutation in other countries.

Mutational ITH in the regional biopsies have been reported in MSI-H CRCs [22-24]. In

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this study, we found mutational ITH of WISP3 in CRCs and added evidence. Surely, there

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might be over- or under-detection of WISP3 frameshift mutation in GCs and CRCs. Exact roles of WISP3 mutational ITH in patients’ diagnosis and therapy as well as in cancer biology should be further investigated.

It remains unclear whether the frameshift mutations play a causal role in tumorigenesis,

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or they reflect the phenomenon that MSI-H tumors have a greater frequency of mutations in many genes. One possible way to address is to examine the survival data to find out if the

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mutation or expression loss of WISP3 is associated with clinical outcome. However, survival

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data of the patients are not available in many patients, especially in the CRCs with ITH due to short follow-up periods after surgeries (less than 5 years). Earlier works suggested a

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possibility of therapeutic targeting for EMT pathway in cancers [1-3, 9]. In this sense, accurate determination of mutation status of related genes is required for such application. Our study shows here that structural alterations by the mutation and additional ITH should be considered before the clinical application to GC and CRC with MSI-H.

Acknowledgements: This study was supported by a grant from National Research Foundation of Korea (2012047939).

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Table 1. Summary of pathologic features of the cancers. MSI-H

MSS/MSI-L

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Feature

Total cases TNM stage I II III IV Location Cecum

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Colorectal carcinomas

25 20

3 31

4 41

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EGC vs. AGC EGC AGC

20 14

15 18 11 1

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I II III IV Lauren’s subtype Diffuse Intestinal

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Total cases TNM stage

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Gastric carcinomas

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15 29 29 3

10 37 30 3

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46 12 2 0

6 6 32 36

CR I

Ascending colon Transverse colon Descending & sigmoid colon Rectum

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EGC: early gastric cancer, AGC: advanced gastric cancer, TNM: tumor, lymph node, metastasis, MSI-H: high microsatellite instability, MSI-L: low microsatellite instability,

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MSS: stable microsatellite instability

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Mutation Location

Wild type

Mutation

WISP3

Exon 5

A9

A8

MSI status of the mutation cases (n)

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MSI-H (11)

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MSI-H: high microsatellite instability

Description

Incidence in MSI-H

Nucleotide change

cancers (%)

(predicted amino acid change)

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Gene

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Table 2. Summary of WISP3 mutation in gastric and colorectal cancers

Gastric: 3/34 (8.8)

Colorectal: 8/76 (10.5)

c.624delA (p.Lys208AsnfsX24)

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WT: wild type

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Table 3. Intratumoral heterogeneity of the WISP3 frameshift mutation in gastric and colorectal cancers

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All cases

Positive WISP3 expression (%)

34 3 31

22 (64.7) 0 (0) 22 (71.0)

76 8 68

51 (67.1) 1 (12.5) 50 (73.5)

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45 0 45

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MSS/MSI-L Gastric cancer Total with WISP3 mutation without WISP3 mutation Colorectal cancer

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Gastric cancer Total with WISP3 mutation without WISP3 mutation Colorectal cancer Total with WISP3 mutation without WISP3 mutation

p-value

CR I

MSI-H

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Table 4. Summary of WISP3 expression and frameshift mutation in gastric and colorectal cancers

38 (84.4) 0 (0) 38 (84.4)

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Total 80 72 (90.0) 1.000 with WISP3 mutation 0 0 (0) without WISP3 mutation 80 72 (90.0) GC: gastric cancer, CRC: colorectal cancer, MSI-H: high microsatellite instability, MSI-L: low microsatellite instability, MSS: stable microsatellite instability

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Figure legend Figure 1. Representative SSCP and DNA sequencings of WISP3 A9 repeat in a colon carcinoma. SSCP (A) and DNA sequencing analysis

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(B) of WISP3 from tumor (Lane T) and normal tissues (Lane N). Direct DNA sequencing analyses (B) show heterozygous A deletion within the

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A9 in tumor tissue as compare to the normal tissue.

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Figure 2. Intratumoral heterogeneity of WISP3 frameshift mutation in colon cancers. A: Direct DNA sequencings show WISP3 c.624delA

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mutation (MT) in a regional biopsy (43-2) and wild-type (WT) WISP3 in the other three regional biopsies (43-1, 43-3 and 43-6). B: Direct DNA

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sequencings show WISP3 c.624delA mutation (MT) in a regional biopsy (45-6) and wild-type (WT) WISP3 in the other six regional biopsies

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(45-1, 45-2, 45-3, 45-4, 45-5 and 45-7).

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Figure 3. Visualization of WISP3 expression in gastric and colorectal cancer tissues by immunohistochemistry. A and D: Normal gastric (A) and colonic (D) mucosal cells show positive WISP3 immunostaining. B and E: Gastric (B) and colon (E) cancers show positive WISP3 immunostaining in the cancer cells. C: In a gastric cancer without the WISP3 frameshift mutation, the cancer cells show no (negative) WISP3 immunostaining. E: In a colon cancer with the WISP3 mutation, the cancer cells show a weak WISP3 (negative) immunostaining.

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Fig. 3