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Expression profiling of cutaneous squamous cell carcinoma with perineural invasion implicates the p53 pathway in the process

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ABSTRACT

Squamous cell carcinoma (SCC) is the second most common cancer worldwide and accounts for approximately 30% of all keratinocyte cancers. The vast majority of cutaneous SCCs of the head and neck (cSCCHN) are readily curable with surgery and/or radiotherapy unless high-risk features are present. Perineural invasion (PNI) is recognized as one of these high-risk features. The molecular changes during clinical PNI in cSCCHN have not been previously investigated. In this study, we assessed the global gene expression differences between cSCCHN with or without incidental or clinical PNI. The results of the analysis showed signatures of gene expression representative of activation of p53 in tumors with PNI compared to tumors without, amongst other alterations. Immunohistochemical staining of p53 showed cSCCHN with clinical PNI to be more likely to exhibit a diffuse over-expression pattern, with no tumors showing normal p53 staining. DNA sequencing of cSCCHN samples with clinical PNI showed no difference in mutation number or position with samples without PNI, however a significant difference was observed in regulators of p53 degradation, stability and activity. Our results therefore suggest that cSCCHN with clinical PNI may be more likely to contain alterations in the p53 pathway, compared to cSCCHN without PNI.

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Differential expression of known p53- or MYC-target genes.Selected known target genes of (a) p53 or (b) MYC were taken from the expression profiling dataset, and plotted as box and whisker (maximum and minimum) plots. Groups are as indicated, showing normalized gene expression (log2) of each of the tumors included in the DASL analysis. Data was analyzed using one-way ANOVA with multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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f2: Differential expression of known p53- or MYC-target genes.Selected known target genes of (a) p53 or (b) MYC were taken from the expression profiling dataset, and plotted as box and whisker (maximum and minimum) plots. Groups are as indicated, showing normalized gene expression (log2) of each of the tumors included in the DASL analysis. Data was analyzed using one-way ANOVA with multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Mentions: To better understand the differences in expression profile between SCC tumors exhibiting no PNI, incidental PNI and clinical PNI, we subjected the lists of genes showing differential expression to pathway analysis. We focused on upstream regulators, specifically transcriptional regulators, of the differentially expressed genes to find potential driver changes. Those with significant z-scores to infer the activation states of predicted transcriptional regulators and significant P-values for overlap of known targets were identified. Genes differently expressed between cSCCHN with clinical PNI and without PNI were significantly associated with apparent activation by the tumor suppressor p53, and repression of Myc when the two groups were compared (Fig. 1c,d; Supplementary Table S5). In addition, an activation associated with NOTCH1 signaling was also identified. Further, cSCCHN with incidental PNI had a signature of gene expression associated with activation of p53 and repression of Myc when compared to cSCCHN without PNI (Supplementary Table S5), although not as significantly as seen with clinical PNI. Comparison of cSCCHN with clinical versus incidental PNI identified a repression of Myc in the clinical PNI samples (Supplementary Table S5), potentially reflecting the more significant pathway repression in these tumors. The list of genes differentially expressed between cSCCHN with clinical PNI and without PNI was additionally subjected to further pathway analysis using KEGG as well as interrogation of potential promoter transcription factor binding sites using TRANSFAC and JASPAR databases, available through Enrichr1718. Both p53 and MYC binding sites were significantly over-represented in the list of differentially expressed genes with the TRANSFAC/JASPAR analysis (Supplementary Table S6). KEGG analysis also identified a significant enrichment of genes identified in the p53 signaling pathway (Supplementary Table S7). Further analysis of the differentially expressed genes showed significantly increased expression of known p53-target genes19, including CDKN1A (p21), BBC3 (PUMA) and TP53I3 (PIG3) in cSCCHN with incidental and /or clinical PNI compared to tumors without PNI (Fig. 2a). There was also significantly decreased expression of well characterized MYC-regulated genes20, including CDK4, ID2, RPL22 and H2AFZ in cSCCHN with clinical and incidental PNI compared to tumors without neural involvement (Fig. 2b).


Expression profiling of cutaneous squamous cell carcinoma with perineural invasion implicates the p53 pathway in the process
Differential expression of known p53- or MYC-target genes.Selected known target genes of (a) p53 or (b) MYC were taken from the expression profiling dataset, and plotted as box and whisker (maximum and minimum) plots. Groups are as indicated, showing normalized gene expression (log2) of each of the tumors included in the DASL analysis. Data was analyzed using one-way ANOVA with multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035993&req=5

f2: Differential expression of known p53- or MYC-target genes.Selected known target genes of (a) p53 or (b) MYC were taken from the expression profiling dataset, and plotted as box and whisker (maximum and minimum) plots. Groups are as indicated, showing normalized gene expression (log2) of each of the tumors included in the DASL analysis. Data was analyzed using one-way ANOVA with multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Mentions: To better understand the differences in expression profile between SCC tumors exhibiting no PNI, incidental PNI and clinical PNI, we subjected the lists of genes showing differential expression to pathway analysis. We focused on upstream regulators, specifically transcriptional regulators, of the differentially expressed genes to find potential driver changes. Those with significant z-scores to infer the activation states of predicted transcriptional regulators and significant P-values for overlap of known targets were identified. Genes differently expressed between cSCCHN with clinical PNI and without PNI were significantly associated with apparent activation by the tumor suppressor p53, and repression of Myc when the two groups were compared (Fig. 1c,d; Supplementary Table S5). In addition, an activation associated with NOTCH1 signaling was also identified. Further, cSCCHN with incidental PNI had a signature of gene expression associated with activation of p53 and repression of Myc when compared to cSCCHN without PNI (Supplementary Table S5), although not as significantly as seen with clinical PNI. Comparison of cSCCHN with clinical versus incidental PNI identified a repression of Myc in the clinical PNI samples (Supplementary Table S5), potentially reflecting the more significant pathway repression in these tumors. The list of genes differentially expressed between cSCCHN with clinical PNI and without PNI was additionally subjected to further pathway analysis using KEGG as well as interrogation of potential promoter transcription factor binding sites using TRANSFAC and JASPAR databases, available through Enrichr1718. Both p53 and MYC binding sites were significantly over-represented in the list of differentially expressed genes with the TRANSFAC/JASPAR analysis (Supplementary Table S6). KEGG analysis also identified a significant enrichment of genes identified in the p53 signaling pathway (Supplementary Table S7). Further analysis of the differentially expressed genes showed significantly increased expression of known p53-target genes19, including CDKN1A (p21), BBC3 (PUMA) and TP53I3 (PIG3) in cSCCHN with incidental and /or clinical PNI compared to tumors without PNI (Fig. 2a). There was also significantly decreased expression of well characterized MYC-regulated genes20, including CDK4, ID2, RPL22 and H2AFZ in cSCCHN with clinical and incidental PNI compared to tumors without neural involvement (Fig. 2b).

View Article: PubMed Central - PubMed

ABSTRACT

Squamous cell carcinoma (SCC) is the second most common cancer worldwide and accounts for approximately 30% of all keratinocyte cancers. The vast majority of cutaneous SCCs of the head and neck (cSCCHN) are readily curable with surgery and/or radiotherapy unless high-risk features are present. Perineural invasion (PNI) is recognized as one of these high-risk features. The molecular changes during clinical PNI in cSCCHN have not been previously investigated. In this study, we assessed the global gene expression differences between cSCCHN with or without incidental or clinical PNI. The results of the analysis showed signatures of gene expression representative of activation of p53 in tumors with PNI compared to tumors without, amongst other alterations. Immunohistochemical staining of p53 showed cSCCHN with clinical PNI to be more likely to exhibit a diffuse over-expression pattern, with no tumors showing normal p53 staining. DNA sequencing of cSCCHN samples with clinical PNI showed no difference in mutation number or position with samples without PNI, however a significant difference was observed in regulators of p53 degradation, stability and activity. Our results therefore suggest that cSCCHN with clinical PNI may be more likely to contain alterations in the p53 pathway, compared to cSCCHN without PNI.

No MeSH data available.


Related in: MedlinePlus