Two critical positions in zinc finger domains are heavily mutated in three human cancer types

A major goal of cancer genomics is to identify somatic mutations that play a role in tumor initiation or progression. Somatic mutations within transcription factors are of particular interest, as gene expression dysregulation is widespread in cancers. The substantial gene expression variation evident across tumors suggests that numerous regulatory factors are likely to be involved and that somatic mutations within them may not occur at high frequencies across patient cohorts, thereby complicating efforts to uncover which ones are cancerrelevant. Here we analyze somatic mutations within the largest family of human transcription factors, namely those that bind DNA via Cys2His2 zinc finger domains. Specifically, to hone in on important mutations within these genes, we aggregated somatic mutations across all of them by their positions within Cys2His2 zinc finger domains. Remarkably, we found that for three classes of cancers profiled by The Cancer Genome Atlas (TCGA)ÐUterine Corpus Endometrial Carcinoma, Colon and Rectal Adenocarcinomas, and Skin Cutaneous Melanoma Ðtwo specific, functionally important positions within zinc finger domains are mutated significantly more often than expected by chance, with alterations in 18%, 10% and 43% of tumors, respectively. Numerous zinc finger genes are affected, with those containing KruÈppel- associated box (KRAB) repressor domains preferentially targeted by these mutations. Further, the genes with these mutations also have high overall missense mutation rates, are expressed at levels comparable to those of known cancer genes, and together have biological process annotations that are consistent with roles in cancers. Altogether, we introduce evidence broadly implicating mutations within a diverse set of zinc finger proteins as relevant for cancer, and propose that they contribute to the widespread transcriptional dysregulation observed in cancer cells

Two critical positions in zinc finger domains are heavily mutated in three human cancer types

<div><p>A major goal of cancer genomics is to identify somatic mutations that play a role in tumor initiation or progression. Somatic mutations within transcription factors are of particular interest, as gene expression dysregulation is widespread in cancers. The substantial gene expression variation evident across tumors suggests that numerous regulatory factors are likely to be involved and that somatic mutations within them may not occur at high frequencies across patient cohorts, thereby complicating efforts to uncover which ones are cancer-relevant. Here we analyze somatic mutations within the largest family of human transcription factors, namely those that bind DNA via Cys2His2 zinc finger domains. Specifically, to hone in on important mutations within these genes, we aggregated somatic mutations across all of them by their positions within Cys2His2 zinc finger domains. Remarkably, we found that for three classes of cancers profiled by The Cancer Genome Atlas (TCGA)—Uterine Corpus Endometrial Carcinoma, Colon and Rectal Adenocarcinomas, and Skin Cutaneous Melanoma—two specific, functionally important positions within zinc finger domains are mutated significantly more often than expected by chance, with alterations in 18%, 10% and 43% of tumors, respectively. Numerous zinc finger genes are affected, with those containing Krüppel-associated box (KRAB) repressor domains preferentially targeted by these mutations. Further, the genes with these mutations also have high overall missense mutation rates, are expressed at levels comparable to those of known cancer genes, and together have biological process annotations that are consistent with roles in cancers. Altogether, we introduce evidence broadly implicating mutations within a diverse set of zinc finger proteins as relevant for cancer, and propose that they contribute to the widespread transcriptional dysregulation observed in cancer cells.</p></div

Massenspektrometrische Präzisionsanalyse eines Antikörper-Bindemotivs in der Linker-Region von C2H2-Zinkfinger-Proteinen: von der Charakterisierung der Epitop-Feinstruktur zur Antikörper-basierten Tumordiagnostik am Beispiel des Endometriumkarzinoms

Mittels ITEM-TWO untersuchte ich, inwieweit eine Tumordiagnostik mit dem anti-HpTGEKP-Antikörper von Aminosäureaustauschen/Phosphorylierungen im bipolaren Antikörperbindemotiv abhängig ist. Getestet wurden 15 Peptidsequenzen: Die 10 häufigsten C2H2-Zinkfinger-Linker und der im Endometriumkarzinom im ZNF331-Gen gehäuft vorkommende H11Y-Austausch, inkl. Phosphorylierungen. Dabei führte die präzisere Differenzierung von Antikörperbindungen zur Einteilung in ‚orthodoxe/unorthodoxe‘ Epitop-Paratop-Bindungstypen, was wegweisend zur Charakterisierung künftiger Antikörperdiagnostika/-therapeutika ist.I applied ITEM-TWO to investigate whether tumor diagnosis with the anti-HpTGEKP antibody depended on amino acid exchanges/phosphorylations within its bipolar binding motif. 15 peptides were tested: The 10 most common C2H2 zinc finger linkers and linkers carrying the H11Y exchange plus phosphorylations, caused by mutation of the ZNF331 gene in uterine corpus endometrial carcinoma. To reach my goal I needed to further differentiate specific antibody binding introducing a novel 'orthodox/unorthodox' binding type, which will improve assessment of future antibody-based diagnostics/therapeutics

Investigating Synovial Sarcoma, X breakpoint proteins in ovarian cancer

Ovarian cancer (OC) affects around 7500 women in the UK every year, but despite this, there is no effective screening strategy or standard treatment. There is a strong correlation between OC prognosis and the stage of diagnosis. If diagnosed during stage I, OC has over a 90% 5-year survival rate, however vague symptoms often lead to late stage diagnosis and a correspondingly poor survival rate. CA-125 is the ‘gold standard’ clinically used serum biomarker for confirming and monitoring OC in patients but is not always present in early stage disease. Previous research has identified expression of the cancer testis antigen SSX2 in stage I and II OC at significantly higher levels than CA- 125. The primary objective of this study was to evaluate patient tumour samples for SSX2 expression alongside other SSX variants, SSX3 and SSX4, to expand investigation into SSX family members as biomarkers for OC. Levels of SSX variants were evaluated for correlations against OC subtype as well as disease stage to acknowledge the diverse histotypes of OC. SSX2 and SSX3 expression was shown to be increased in early stage OC when compared to normal adjacent tissue. Metastatic OC was also found to significantly express SSX2, suggesting that SSX3 may be a superior early stage biomarker candidate. Collectively this data revealed variations in the level of SSX expression across OC stages and histotypes. Following successful development of in vitro overexpression models of SSX family members, global transcriptomic profiling was used to identify novel transcripts and pathways altered by SSX2A, SSX2B and SSX3. Transcriptional profiling of each overexpression model highlighted that SSX variants modulate unique and common downstream targets and resulting functions. This work suggested that SSX2A, SSX2B and SSX3, alone or in combination, appear to contribute to cancer progression and clinical outcomes. Consequently, in vitro and in vivo studies were performed to assess the impact of each SSX family member on cell proliferation and motility in OC. All SSX variants in this study, SSX2A, SSX2B, SSX3 and SSX4 significantly promoted cell proliferation in an OC cell line. Changes were also seen in key epithelial to mesenchymal transition markers, including an increase in the expression of transcription factor SLUG. Further, novel tumour xenograft models developed in this study highlighted that SSX3 overexpression increased OC proliferation in vivo. Overall, these results highlight the importance of investigating SSX family members in OC, as biomarkers and promoters of cancer development