Novel oncogenic transcriptional targets of mutant p53 in esophageal squamous cell carcinoma

Missense mutations in the DNA binding domain of p53 are observed frequently in esophageal squamous cell carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common “hotspot” p53 mutations while rarer mutations are poorly characterized. In this study, we report the characterization of rare, “non‐hotspot” p53 mutations from ESCC. In vitro tumorigenic assays performed following ectopic‐expression of certain “non‐hotspot” mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome‐wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harboring mutant p53. Of these, ARF6, C1QBP, and TRIM23 were studied further. Reverse transcription‐quantitative PCR (RT‐qPCR) performed on RNA isolated from ESCC tumors revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild‐type and several mutant p53 forms followed by RT‐qPCR, chromatin affinity‐purification (ChAP), and promoter‐luciferase assays indicated the exclusive recruitment of p53 mutants—P190T and P278L, to the target genes leading to the activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effects of the genes were confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of “non‐hotspot” mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor‐specific p53 mutations.

In India, cancer of the esophagus holds the eighth and sixth positions in terms of incidence and mortality rate among all cancers, respectively. 1Factors contributing to esophageal cancer development include genetic predisposition, age, excessive smoking and alcohol use, poor diet, obesity, Human Papillomavirus (HPV) infection, and medical conditions like Barrett's esophagus. 2TP53 is the most frequently mutated gene in esophageal cancers. 3It encodes a transcription factor involved in the regulation of several cardinal cell processes including cell cycle, DNA damage repair, metabolism, senescence, cell death, and so forth.Somatic alterations in TP53 that disrupt the tumor-suppressor activities of the protein are observed in 50% of all cancers.Nearly 90% of missense mutations, the most predominant type of alterations observed in TP53, occur in the sequence encoding the central DNA-binding domain. 4,5In cancers, p53 is recurrently altered at six amino acid positions namely R175, G245, R248, R249, R273, and R282, which account for approximately a quarter of all reported mutations (referred to as "hotspot" mutations). 6,7In addition to the inactivation of tumor-suppressor functions, these "hotspot" mutant p53 proteins induce additional pro-oncogenic phenotypes and are thus termed "gain-of-function" mutants. 8Mutant p53 proteins exhibit gain-of-function properties through various means, of which transcriptional activation of oncogenes appears to be the most frequent. 9,10In-depth studies assessing the oncogenic effects of mutations in TP53 are however generally restricted to the hotspot mutations while rarer mutations remain poorly characterized.
In this study, we performed genome-wide transcript analysis of esophageal squamous cell carcinoma (ESCC) tumors stratified by p53 status and identified three novel oncogenic transcriptional targets of "non-hotspot" mutant p53-ARF6, C1QBP, and TRIM23.

| Patient samples and immunohistochemistry (IHC)
The ESCC tumors along with their corresponding matched normal samples were collected from various hospitals in Hyderabad.The categorization of tumors based on the nuclear-stabilization status of p53 was performed as detailed in our previous study. 11Samples exhibiting nuclear stabilization of the p53 protein were termed NS+ (nuclear-stabilized), a proxy for missense mutations in TP53, whereas those lacking stabilized p53 were termed NS− (nuclear-unstable).Formalin-fixed paraffin-embedded blocks containing tumor or matched normal patient samples were cut into sections of 4 μm thickness using a microtome (HistoCore RM2125 RTS) (Leica).The tissue sections were processed as previously described. 12The specimens were probed with specific primary antibodies (anti-p53 [EMD Millipore; Calbiochem], anti-ARF6 [Abcam], anti-C1QBP [Abcam], anti-TRIM23 [Abcam]) as described earlier. 13The IHC stained slides were evaluated and scored by two experienced pathologists blinded to the clinical and molecular information of the samples.For ARF6, the slides were scored for cytoplasmic and membrane staining as 0: negative; 1: weak staining; 2: moderate staining; 3: strong staining.For C1QBP and TRIM23, the slides were scored based on the number of positively stained cells as 0: negative; 1: <25%; 2: 25%−50%; 3: 50% −75%; 4: >75%, as well as intensity of staining as 0: negative; 1: weak staining; 2: moderate staining; 3: strong staining.The summation of both scores yielded the final scores.Tumor DNA corresponding to TP53 exons 2−9 were screened for mutations by Sanger sequencing as described earlier. 14

| Genome-wide transcript profiling
Thirty-six ESCC tumors (19 NS− and 17 NS+) were selected for gene-expression analysis ensuring no significant variability in several parameters including patient age and sex, tumor grade and stage, status of tobacco or alcohol use, as well as molecular variables such as EGFR, FHIT, microsatellite instability, and so forth.Tumor samples infected with HPV were excluded from the study.Microarray based gene expression analyses were performed as described in our previous study. 15The transcript levels of genes were also analyzed using reverse transcription-quantitative PCR (RT-qPCR) to confirm that the microarray expression profiles faithfully reflected the expression status of the genes.The microarray data have been deposited in the Gene Expression Omnibus (GSE218109).

| Assessment of frequency of nonhotspot mutations in cancer databases
The frequencies of the non-hotspot and hotspot TP53 mutations identified in ESCC tumors in the present study were measured and compared to their frequencies in three standard cancer databases-The Cancer Genome Atlas, Catalogue of Somatic Mutations in Cancer, and The TP53 database.

| Molecular genetic manipulations
For overexpression studies, wild-type TP53 cDNA was amplified from p53-GFP plasmid (Addgene) and cloned into the pFN21A HaloTag® CMV Flexi® Vector (Promega Corporation) vector using a restriction digestion and ligation method.The missense mutant constructs were generated through PCR-based site directed mutagenesis (SDM).For immunofluorescence studies, pEGFP-N1 (Addgene), wild-type p53-EGFP (Addgene) plasmid, and p53-EGFP mutant constructs generated through SDM were employed.Promoter fragments corresponding to the wild type and mutant p53 target genes were amplified by PCR using DNA from healthy donors and cloned into the pGL4.26[luc2minP Hygro] vector (Promega Corporation) for promoter-luciferase assays.p53-pFN21A expression constructs were transfected into cell lines using Lipofectamine™ 2000 Transfection Reagent (Invitrogen).
The short-hairpin loop RNAs (shRNAs) targeting ARF6, C1QBP, and TRIM23 were selected from the RNAi consortium shRNA library hosted by the Broad Institute (ARF6: TRCN0000294069, TRCN0000305862, TRCN0000 379678, TRCN0000379907, and TRCN0000305860, C1QBP: TRCN0000057106, TRCN0000057104, TRCN0000 370165, TRCN0000057107, and TRCN0000370164, and TRIM23: TRCN0000233071, TRCN0000233067, TRCN00 00233068, TRCN0000233070, and TRCN0000233069).Non-targeting scramble shRNA (Addgene) which was employed as a control and the shRNAs targeting the three genes were cloned in the pLKO.1 puro vector (Addgene) for viral particle generation in HEK293T cells.shRNA stable cell lines were generated as described earlier. 11or rescue experiments, the coding sequences of ARF6, C1QBP, and TRIM23 were amplified from their respective pLenti6.3/V5DEST vectors (DNASU; Arizona State University) and cloned into the pEGFP-N1 vector by restriction digestion.Further, synonymous mutations in the region encoding three consecutive amino acids corresponding to the shRNA seed region in each of the three constructs were generated (to prevent/reduce the degradation of the target gene transcripts under the influence of shRNA) through SDM.

| Assessment of the subcellular localization of p53-EGFP proteins
H1299 cells were transfected with the pEGFP vector or p53 wild-type/mutant-EGFP constructs and incubated for 36 h.The slides were washed with 1X PBS, fixed for 10 min with 4% paraformaldehyde and subsequently washed thrice with 1XPBS.Five percent bovine serum albumin was added for blocking and the slides were incubated for an hour on a rocker at room temperature.The slides were washed thrice with 1X PBS, then mounted with VECTASHIELD® Antifade Mounting Medium with DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories) and sealed, followed by incubation in the dark for 30 min.The slides were visualized under 65X magnification in the Leica SP8 (Leica Microsystems) confocal microscope.The localization of each taggedprotein was assessed with DAPI as a reference for the nucleus.

| Dual-luciferase reporter and chromatin affinity purification (ChAP) assays
Luciferase reporter assays were performed similar to our previous study. 15ChAP assays were performed following transfection with various p53 expression constructs, to evaluate recruitment of mutant p53 to the endogenous promoter regions of the three target genes, using the HaloCHIP™ System (Promega Corporation) according to the manufacturer's protocol. 11The region evaluated for each gene in the ChAP experiment included the region analyzed in the promoter-luciferase assays.Promoter enrichment after affinity purification was measured by RT-qPCR using a standard protocol. 11,159 | In vivo tumorigenesis assays 5 × 10 6 AW13516 cells stably expressing either scrambled shRNA or shRNAs targeting C1QBP or TRIM23 were mixed with 100 μL of 1XPBS and 100 μL of Matrigel (Corning) and injected into the flanks of 5−8 weeks old FOXN1 −/− nude mice (seven and nine mice per batch for C1QBP and TRIM23, respectively).16 The dimensions of the tumors were measured at weekly intervals for a period of 8 weeks post-injection.The mice were then necropsied and the tumor sizes and weights were measured.

| Genome-wide transcriptome analysis of ESCC tumors reveals novel transcriptional targets of mutant TP53
To identify the transcriptional targets of ESCC-specific p53 mutants, microarray-based transcript profiling was performed on a cohort of 19 p53 nuclear negative (NS−) and 17 p53 nuclear positive (NS+) ESCC samples (Supporting Information S1: Table 1).Following computational analysis of the data, several gene-sets differentially enriched in p53 NS+ tumors were identified.The top 40 differentially enriched gene-sets (p < 0.0007) represented several protumorigenic processes including p53 regulated hypoxia pathway, Wnt signaling, Myc, ERBB/EGFR signaling, and so forth (Supporting Information S1: Figure 1A).
Application of Significance Analysis of Microarrays on all 1024 genes that constituted the 40 gene-sets, revealed seven genes that were significantly upregulated in NS+ samples (Supporting Information S2: Figures 1B-C).The transcript levels of TP53 itself were significantly upregulated in NS+ samples (Supporting Information S2: Figure 1B), validating our previous results obtained for squamous cell carcinoma of the oral tongue. 11Among the genes that appeared to be differentially upregulated in NS + samples ARF6, C1QBP, and TRIM23 were chosen for further studies based on their purported role in cancer.Transcript levels of the three genes were significantly higher in NS+ samples as confirmed through RT-qPCR (Figure 1A and Supporting Information S1: Table 2A).The expression levels of the three genes significantly correlated with the transcript levels of TP53 (Figure 1B and Supporting Information S1: Table 2B).
We identified 11 TP53 mutations in the ESCC NS+ samples subjected to gene expression analysis (Supporting Information S1: Table 1 and Figure 1D).No TP53 mutation was detected in the ESCC NS− samples, as expected.Several of these mutations exhibited significantly low frequencies in cancer mutation databases (Supporting Information S1: Table 3).We confirmed the differential expression of the three putative target genes in p53 mutant and wild-type samples (Figure 1A and Supporting Information S1: Table 2C).Of the various p53 mutants detected in the ESCC tumor samples, R158P, P190T, Y236C, V272M (identified in a non-arrayed ESCC sample) and P278L (termed "non-hotspot" mutations) were further assessed for their oncogenic potential in H1299; three highly oncogenic hotspot mutant proteins namely R175H, R248W (also detected in the ESCC tumor samples; Supporting Information S1: Table 1), and R273H were employed as controls.All the p53 mutant forms predominantly exhibited nuclear localization (Supporting Information S1: Figure 2).The hotspot p53 mutants supported significantly elevated cell viability/proliferation as well as migration potential whereas, the wild-type p53 supported significantly lower activities in the same assays (Figure 2A,B). 17The non-hotspot mutants P190T and P278L consistently supported significantly high cell viability/proliferation and migratory activity (Figure 2A,B).
Based on the above results, we proceeded to scrutinize the activity of P190T and P278L in additional squamous cancer cell lines namely NT-8e and JHU-011 to validate the results obtained in H1299.P190T and P278L mutants supported significant induction of tumorigenic activity in both cell lines, similar to the hotspot mutants (Supporting Information S1: Figures 3 and 4).Finally, we evaluated the tumorigenic potential of P190T and P278L in comparison to other non-hotspot and hotspot mutations in the esophageal cancer cell line KYSE-410.Both P190T and P278L induced tumorigenic Since TP53 hotspot mutations exert their protumorigenic function via non-canonical transcriptional activation of oncogenes 18 ; we endeavored to determine whether the oncogenic potential exhibited by P190T and P278L mutants were via the transcriptional activation of ARF6, C1QBP, and TRIM23.We thus performed ectopic expression of wild-type and several mutant p53 forms and evaluated the transcriptional activation of ARF6, C1QBP, and TRIM23 in H1299, AW13516, and KYSE-410.CDKN1A and MVK, transcriptional targets of wild-type and hotspot mutant p53 forms, respectively, were evaluated as controls.RT-qPCR analyses revealed that only P190T and P278L consistently activated the expression of the target genes (Figure 3A−C).CDKN1A and MVK were exclusively activated by the wild type and hotspot mutant p53 forms, respectively.We also evaluated the ability of various p53 proteins to activate the promoters of the three target genes in H1299 and KYSE-410.P190T and P278L were able to increase the activity of ARF6, C1QBP, and TRIM23 promoters (Figure 3D,E).
Wild-type p53 induced the expression of the luciferase gene linked to the ATF3 promoter, while the MVK promoter-linked luciferase expression was regulated by the hotspot mutant R248W (Figure 3D,E).Finally, ChAP assay was performed in H1299 cells to assess whether the regulation of the target genes occurred due to the recruitment of p53 to the respective promoters.Upon their ectopic expression, P190T and P278L mutant p53 forms were recruited to the three endogenous target gene promoters (Figure 3F); a similar experiment targeting the gene body region of the three genes revealed a markedly lower enrichment (Supporting Information S1: Figure 5).

| The transcriptional targets of nonhotspot mutant p53 play oncogenic roles in squamous cell carcinoma cell lines
Given the ability of mutant p53 to induce oncogenic functions in tumor cells via transcriptional activation of relevant genes, we proceeded to determine if ARF6, C1QBP, and TRIM23 could exhibit pro-oncogenic functions in squamous cancer cell lines.We thus performed shRNA-based knockdown studies in the KYSE-410 (Figure 4A−E and Supporting Information S1: 6), AW13516 and H1299 cells (Supporting Information S2: Figures 6 and 7).Knockdown of ARF6, C1QBP, and TRIM23 resulted in a reduction in several tumorigenic properties assessed by evaluating cell viability, cell growth, colony formation, and migratory potential (Figure 4A−E and Supporting Information S1: 7).Overexpression of the respective proteins resulted in a significant rescue of the original tumorigenic properties of the cell lines, thus confirming the specificity of results obtained with knockdown of each of the three genes (Figure 5, Supporting Information S1: 6, 8 and 9).Interestingly, ectopic expression in control cells (generated from scramble shRNA) also revealed activation of tumorigenic properties further confirming the potential oncogenic roles of ARF6, C1QBP, and TRIM23 (Figure 5, Supporting Information S1: 6, 8 and 9).
Next, we evaluated the oncogenic potential of C1QBP and TRIM23 in nude mice xenograft models.AW13516 cells exhibiting a knockdown of each gene resulted in significant reduction in tumor size and volume in comparison to the control cells (Figure 4F−K and Supporting Information S1: 10).
We assessed the ARF6, C1QBP, and TRIM23 transcript levels in ESCC tumors compared to normal samples.As expected, the transcript levels of all three genes were significantly higher in the tumors in comparison to the matched normal samples, further validating the potentially oncogenic nature of these genes (Figure 1C; Supporting Information S1: Table 2D).
The protein expression and localization of p53 and its target gene products was determined by IHC on ESCC tumor samples (Figure 1D).A positive correlation was observed between the nuclear stabilization of p53 and the expression of all three mutant p53 transcriptional target proteins (Figure 1D and Supporting Information S1: Table 4).These findings suggest a clinical relevance of the discovery of novel transcriptional targets of mutant p53.

| DISCUSSION
Several recent studies have revealed the oncogenic potential of p53 "hotspot" mutant forms in various cancers.The oncogenic potential exhibited by mutants such as R175H, R273H, and R248Q/W are a result of their ability to transcriptionally activate genes with the ability to induce oncogenic changes in cells. 8It has also been shown that mutant p53 and hypoxia play important roles in altering metabolic pathways in hepatocellular carcinoma leading to alterations in the tumor microenvironment and immune pattern encouraging cancer development, and can thus be adopted as potential targets for cancer therapy. 19Similarly, another study reported the involvement of p53-G245S in the induction of hnRNPA2B1-AGAP1-mediated exosome formation that promotes ESCC progression. 20P53-R175H in association with periostin was shown to activate STAT1 and its transcriptional targets in the esophageal cancer tumor microenvironment enabling the invasion of esophageal epithelial cells into the extracellular matrix. 21Evaluation of the potential oncogenic roles of less-frequent nonhotspot p53 mutants is however lacking.The main objective of the current study was therefore to evaluate the oncogenic potential of these rare p53 mutant forms identified from ESCC tumors and to characterize the transcriptional networks they may regulate.In our study, P190T and P278L exhibited robust oncogenic functions.
Based on a comprehensive analysis, we identified three potential oncogenic transcriptional targets (ARF6, F I G U R E 4 ARF6, C1QBP, TRIM23 exhibit oncogenic properties determined by performing various tumorigenic assays following stable knockdown.Tumorigenic assays following stable knockdown of each of the three genes in KYSE-410 cells is shown (A−E).MTT (A−C); cell migration (D); colony formation (E).Each result is based on at least three independent experiments; *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student's t-test).Validation of the oncogenic role of C1QBP (F, H, and J) and TRIM23 (G, I, and K) in AW13516 squamous carcinoma cells based on nude mice xenograft assays.Representative images of tumors excised from the animals (F, G) and quantitation of their volume and weight (H, I).The periodic change in tumor volume is shown in (J, K). **p < 0.01; ****p < 0.0001 (unpaired Student's t-test).C1QBP, and TRIM23) of rare p53 mutant forms (P190T and P278L) with high relevance to ESCC.The ARF6 protein controls cell adhesion, actin remodeling, cytokinesis and membrane trafficking. 22,23High levels of ARF6 and its downstream targets have been reported in cancers of the breast, pancreas, liver, and lung, and have been linked to elevated invasive, proliferative and metastatic potential of the tumors. 24,25ARF6 and its effector protein AMAP1 form the EGFR-GEP100-ARF6-AMAP1 axis to induce invasion in various cancers 26 besides disturbing the cell adhesion regulated by Ecadherin. 27High levels of APE1 were found to increase the oncogenic potential in esophageal adenocarcinoma tumors and cell lines through an APE1-ARF6-MMP-14 signaling axis. 28Mutant p53 is also reported to cause the elevation of ARF6 levels which in turn activate the mevalonate pathway that induces metastasis. 29These two ARF6-mediated tumorigenesis pathways may potentially also be functional in ESCC tumors.C1QBP levels are elevated in cancers of the breast, prostate, thyroid, esophagus, endometrium, pancreas, stomach, colon, and lung. 30,31C1QBP levels have been found to exhibit a strong correlation with metastasis and high tumor stage in breast cancer, and are associated with higher stage and recurrence in prostate cancer. 32,33C1QBP negatively regulates the transcriptional activation of wild-type p53 by binding to its tetramerization domain and blocking its oligomerization resulting in its increased nuclear export followed by destruction. 34C1QBP interacts with RPS15 partner, a pro-oncogenic protein that is elevated in ESCC tumors, and has been shown to promote proliferation and metastasis of esophageal cancer cell lines. 35This may be a potential mechanism of tumorigenesis in ESCC which may be relevant in our patient cohort.TRIM23 is an E3 ubiquitin ligase that localizes to lysosomes and golgi vesicles and plays a role in vesicular transport and phospholipase-D activation. 36TRIM23 transcript levels were found to be high in cancers of the stomach, liver, lung, and colorectum. 37,38TRIM23 activates NF-κB in hepatocellular carcinoma cells leading to the suppression of HNF-1α and in turn of miR-194 resulting in increased invasion and migration. 39The E3 ubiquitin ligase activity of TRIM23 is hypothesized to lead to a reduction in wild type p53 protein levels in colorectal cancer (CRC) cells, and induce proliferation of CRC cell lines. 37This feature of TRIM23 may be also be responsible for encouraging oncogenesis in ESCC tumors and cell lines.
Therefore, in this study, we identified P278L and P190T as novel gain of function non-hotspot p53 mutations presumably involved in ESCC tumorigenesis.Three novel transcriptional targets namely ARF6, C1QBP, and TRIM23 of P278L and P190T were identified which exhibit pro-oncogenic properties indicating their direct or indirect role in the development of squamous cell carcinomas.It is essential to define the complete transcriptional network of different p53 mutants which is expected to aid in the development of suitable therapeutic regimes to efficiently treat cancer patients.We believe that the work done in this study is an important step in that direction.

F
I G U R E 1 Validation of novel transcriptional targets of non-hotspot mutant p53 forms.(A) Relative transcript levels of the target genes in ESCC tumors stratified for p53 nuclear stabilization or mutation status.(B) Correlation of transcript levels of the three genes with that of TP53 in ESCC tumor samples.*p < 0.05; **p < 0.01; ****p < 0.0001; (unpaired Student's t-test).(C) Quantitation of relative transcript levels of the three genes in tumor versus normal ESCC samples.(D) Correlation of expression of the three genes with that of p53 using IHC on an ESCC tissue microarray.ESCC, esophageal squamous cell carcinoma; NS, nuclear stabilization.activity as evidenced from the MTT, cell growth, and cell migration assays (Figure 2C−E).

F I G U R E 2
Evaluation of oncogenic potential of "non-hotspot" mutant p53 proteins.MTT (A and C), migration (B and E), and growth (D) assays performed in H1299 (A and B) and KYSE-410 (C−E) cells.Statistical significance is calculated on the difference between each p53 construct and vector based on at least three independent experiments; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant (unpaired Student's t-test).V, expression vector.

F
I G U R E 5 Rescue of reduced cell viability (A−C), cell growth (D), and migration (E, F) upon respective protein expression in KYSE-410 cells harboring stable knockdown of ARF6, C1QBP, or TRIM23.Rescue is obtained upon ectopic expression of both wild-type and mutant (generated to negate effect of shRNA) proteins.Each result is based on at least three independent experiments; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant (unpaired Student's t-test).