SMGs
We next applied the MutSigCV method36 to identify SMGs in the combined 192 ESCC tumors (Figure S1E) and discovered nine such genes driven by point mutations (false discovery rate, q < 0.1) and six further genes with p < 0.01 (Figure 3A). Eleven of these 15 genes—including AJUBA, ZNF750, FAT1, FBXW7 (MIM 606278), and PTCH1 (MIM 601309) and the chromatin-remodeling genes CREBBP (MIM 600140) and BAP1 (MIM 603089)—harbored frequent inactivating mutations. Although AJUBA, ZNF750, FAT1, and FBXW7 were recently implicated as tumor suppressors in ESCC,7,37 their roles in mice models and the mechanisms by which they function as tumor suppressors are limited. As in other cancers,38,39 particularly EAC,19 this analysis also identified well-known cancer-associated genes, such as TP53 (MIM 191170), PIK3CA, and CDKN2A (MIM 600160), as SMGs in ESCC, thus providing evidence of common dysfunctions in cell-cycle control and apoptotic signaling.
AJUBA, which encodes a LIM domain protein, inhibits the ATR-dependent DNA-damage response and is involved in several cellular processes, such as cell-fate determination, cytoskeletal organization, transcriptional repression, mitotic commitment, cell-cell adhesion, and migration.40 Along with LIMD1 (MIM 604543), AJUBA has been proposed to be a major component of the miRNA-mediated gene-silencing machinery and might have a tumor-suppressive function.41 However, the biological functions of AJUBA in ESCC tumorigenesis have never been reported. The following AJUBA mutations were identified and verified in four tumor samples: two stop-gain mutations (c.985G>T [p.Glu329∗] and c.1057C>T [p.Gln353∗]) and two frameshift indels (c.790_791insT [p.Val264fs∗] and c.152delG [p.Gly51fs∗]) in the 104-individual cohort and one frameshift insertion (c.1249_1250insA [p.Ala417fs∗]) and one splice-site mutation in two individuals from our previous cohort6 (Figure 3B; Figure S7A; Table S3). These mutational events result in truncated or disrupted protein products that lack proper LIM domains, indicating that they are loss-of-function mutations, and these mutations in AJUBA, encoding the truncated or disrupted protein, are expected to promote ESCC oncogenesis. In support of this possibility, immunoblot analysis showed the presence of truncated AJUBA in ESCC tumors (Figure 4A). AJUBA knockdown in KYSE140 and KYSE510 cells led to increased cell growth, colony formation, cell migration, and cell invasion (Figure S8). Moreover, exogenous levels of wild-type AJUBA in KYSE30 and KYSE150 cells with low endogenous AJUBA levels suppressed cell growth, cell migration, and cell invasion; these effects were abrogated by AJUBA alterations (p.Gln353∗ and p.Val264fs∗; Figures 4B–4F). Together with our genetic observations, these functional data indicate that AJUBA might act as a tumor suppressor in ESCC and that the AJUBA mutations observed in ESCC abrogate its tumor-suppressive function.
Mutations in ZNF750, a tumor-associated gene located at 17q25,7 were identified in 6% of ESCC tumors. We identified 11 somatic mutations, of which we verified (via Sanger sequencing) six, including two nonsense mutations (c.620G>A [p.Trp207∗] and c.209C>A [p.Ser70∗]), one indel (c.108_111del [p.Glu37Lysfs∗]), and three missense mutations (c.96T>A [p.Phe32Leu], c.1520A>C [p.Asp507Ala], and c.1508C>G [p.Ser503Cys]) in ZNF750 in the 104-individual cohort. In addition, five (c.414C>A [p.Cys138∗], c.770C>A [p.Ser257∗], c.85C>T [p.Gln29∗], c.625_626insAA [p.Ala209fs∗], and c.1621G>A [p.Ala541Thr]) out of 11 somatic mutations were verified by Sanger sequencing in our previous cohort6 (n = 88; Figure 3B). Sixty-four percent of the mutations identified in ZNF750 are inactivating. Additionally, ZNF750 deletions, but no somatic mutations, were observed in 3 out of 14 tumor samples in the WGS set (21%, G-score > 0.23) and validated with a qPCR copy-number assay. The qPCR copy-number assay also determined that four out of six tumor samples harboring ZNF750 mutations or indels in the 90-sample WES set were affected by deletions, indicating that inactivation of both alleles had occurred in these individuals (Figures S9A–S9C). Notably, frequent loss of heterozygosity at 17q25.3 has been previously reported in ESCC,42 but no gene at or near the 17q25.3 region was identified. Our genetic data strongly indicate that ZNF750 is the missing piece of this puzzle.
We next used the immunohistochemical method to determine whether a correlation exists between these genetic changes and the amount of ZNF750. As expected, ZNF750, a nuclear factor, was strongly stained in the nuclei of normal esophageal epithelial cells. Surprisingly, though, it was dramatically upregulated in the cytoplasm of ESCC tumor cells in comparison to that of normal tissue cells. Moreover, individuals with tumors harboring ZNF750 mutations had higher cytoplasmic expression levels than did those lacking ZNF750 mutations (Figure 5A). Mislocalization of a truncated (p.Ser70∗) ZNF750 (Figure 5B) indicated that cytoplasm translocation was partly caused by loss of the C-terminal nuclear localization sequence. ZNF750 regulates the gene program controlling terminal epidermal differentiation.43 For investigating a function for ZNF750 in tumorigenesis, shRNA-mediated stable ZNF750 depletion was followed by transfection with wild-type or altered (p.Ser70∗ or p.Trp207∗) ZNF750 in KYSE150 and KYSE140 cells. ZNF750 knockdown strongly promoted KYSE150 and KYSE140 cell proliferation, migration, and invasion. Moreover, functional studies demonstrated that wild-type ZNF750 inhibited cell growth, migration, and invasion, and this effect was abrogated by altered (p.Ser70∗ or p.Trp207∗) ZNF750 (Figure 5C; Figures S9D and S9E). Finally, ZNF750 depletion and p.Ser70∗ ZNF750 markedly increased tumor size in the xenograft system in mice, whereas wild-type ZNF750 significantly decreased it (Figure 5D). Our genetic observations and functional data therefore suggest that ZNF750 acts as a tumor suppressor in ESCC.
FAT1, which encodes a cadherin-like protein commonly expressed in epithelial tissues,44 was mutated in 15% of ESCC tumors. Notably, 13 mutations (77%) were truncating (stop-gain and frameshift) (Table S3). FAT1 is reported to regulate cell-cell adhesion and other cell behavior by controlling actin polymerization.44 Recently, Morris et al. reported that FAT1 suppresses cancer cell growth by binding β-catenin and preventing nuclear localization.44 FAT1 inactivation, via mutations that affect the cytoplasmic domain, leads to aberrant Wnt/β-catenin signaling in multiple cancer types.45 In our cohort, we discovered mutations affecting cadherin repeats and laminin G domains but no mutations affecting the cytoplasmic domain. This observation indicates that mutations affecting FAT1 extracellular domains might disrupt cell-cell associations and increase invasiveness and thus potentially contribute to ESCC tumorigenesis. FBXW7 mutations were observed in eight ESCC tumors in our cohort: these included two nonsense mutations (c.1005T>A [p.Cys335∗] and c.409G>T [p.Glu137∗]), one frameshift deletion (c.736_739del [p.Gly247Profs∗] and inactivating mutations), and five missense mutations predicted to be deleterious by SIFT and PolyPhen-2 analyses (Table S3).46 FBXW7 mutations and copy-number loss and a subsequent decreased FBXW7 level have been observed in various cancer types.46,47 Decreased amounts of FBXW7 are reported to correlate with poor prognosis;47 however, we observed no correlation between FBXW7 mutations and prognosis in our cohort.
Inactivating mutations in several chromatin-remodeling genes, including CREBBP and BAP1, frequently occurred in our 104 ESCC samples. CREBBP and EP300 (MIM 602700) mutations occurred in 11/104 ESCC tumors (Table S3). Inactivating CREBBP and EP300 mutations have been reported in various human cancer types.6,48 We also identified mutations in BAP1, which encodes a nuclear deubiquitinase involved in chromatin remodeling.49 BAP1 mutations have been reported in renal carcinoma and uveal melanoma,49 but not in ESCC to date. Moreover, frequent truncating mutations were observed in the chromatin-remodeling genes KMT2D (MLL2 [MIM 602113]) and KMT2C (MLL3 [MIM 606833]) (14% together) and KDM6A (MIM 300128) (3%) (Table S3). At least one chromatin-remodeling gene was altered in 33/104 ESCC tumors.