Increased Cycling Cell Numbers and Stem Cell Associated Proteins as Potential Biomarkers for High Grade Human Papillomavirus+ve Pre-Neoplastic Cervical Disease

High risk (oncogenic) human papillomavirus (HPV) infection causes cervical cancer. Infections are common but most clear naturally. Persistent infection can progress to cancer. Pre-neoplastic disease (cervical intraepithelial neoplasia/CIN) is classified by histology (CIN1-3) according to severity. Cervical abnormalities are screened for by cytology and/or detection of high risk HPV but both methods are imperfect for prediction of which women need treatment. There is a need to understand the host virus interactions that lead to different disease outcomes and to develop biomarker tests for accurate triage of infected women. As cancer is increasingly presumed to develop from proliferative, tumour initiating, cancer stem cells (CSCs), and as other oncogenic viruses induce stem cell associated gene expression, we evaluated whether presence of mRNA (detected by qRT-PCR) or proteins (detected by flow cytometry and antibody based proteomic microarray) from stem cell associated genes and/or increased cell proliferation (detected by flow cytometry) could be detected in well-characterised, routinely collected cervical samples from high risk HPV+ve women. Both cytology and histology results were available for most samples with moderate to high grade abnormality. We found that stem cell associated proteins including human chorionic gonadotropin, the oncogene TP63 and the transcription factor SOX2 were upregulated in samples from women with CIN3 and that the stem cell related, cell surface, protein podocalyxin was detectable on cells in samples from a subset of women with CIN3. SOX2, TP63 and human gonadotrophin mRNAs were upregulated in high grade disease. Immunohistochemistry showed that SOX2 and TP63 proteins clearly delineated tumour cells in invasive squamous cervical cancer. Samples from women with CIN3 showed increased proliferating cells. We believe that these markers may be of use to develop triage tests for women with high grade cervical abnormality to distinguish those who may progress to cancer from those who may be treated more conservatively

The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension

Abstract Human WIPI β-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan

The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension

Human WIPI beta-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan.Peer reviewe

Proteomic array detection of stem cell associated proteins in pooled aliquots from 9 HR-HPV+ve cervical samples from women with CIN3 (A) and 9 HPV –ve samples with normal cytology (B); (C & D) map and key for array spots; (E) average pixel density ranked by ratio of pooled CIN3:normal samples for each protein on the array; (F) sox2, TP63 and HCG mRNAs are upregulated in HPV+ve cervical samples with severe dyskaryosis compared to HPV–ve samples with normal morphology (* = p<0.05, ** p<0.01, Mann Whitney test).

<p>Proteomic array detection of stem cell associated proteins in pooled aliquots from 9 HR-HPV+ve cervical samples from women with CIN3 (A) and 9 HPV –ve samples with normal cytology (B); (C & D) map and key for array spots; (E) average pixel density ranked by ratio of pooled CIN3:normal samples for each protein on the array; (F) sox2, TP63 and HCG mRNAs are upregulated in HPV+ve cervical samples with severe dyskaryosis compared to HPV–ve samples with normal morphology (* = p<0.05, ** p<0.01, Mann Whitney test).</p

TP63 and SOX2 staining in cervical biopsies.

<p>Immunohistochemical staining of cervical biopsies. Bars  =  50µm.(A) normal cervix no primary control; (B) normal cervix stained with anti-SOX2; (C) Squamous cell cervical carcinoma no primary control; (D) representative TP63, and (E) representative SOX2, staining of tumour cells. For both TP63 and SOX2 staining was seen in the nucleus of positive cells (examples indicated by solid arrows); negative cells were a minority of tumour cells (examples indicated by unfilled arrows). (F) Image analysis results of % nuclear +ve tumour cells in biopsies. Parallel sections from 11 cases were stained with SOX2 and TP63. Tumour cells were evaluated for their nuclear expression of the transcription factors. There was no significant difference between the data for SOX2 and TP63 (Wilcoxon signed rank test).</p

Flow cytometric detection of cycling cells in cervical samples.

<p>(A) LBC samples stratified by HPV status and histology; samples from women with CIN3 are significantly different from samples with normal cytology and from CIN1 and CIN2, 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus CIN3 group); (B) LBC samples stratified by HPV status and cytology results only; samples with severe dyskaryosis are significantly different from all normal samples 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus severe disease group). * p = <0.05, ** p = <0.01, *** p = <0.001.</p

Samples with increased TRA-1-60+ve cells from patients with CIN3 have enhanced expression of stem cell proteins.

<p>A) Flow cytometric analysis of samples used for proteomic microarray. Dotted lines show that the three samples with a significant increase in TRA-1-60 +ve cells have similar levels of cycling cells to the other three CIN3 samples. *  = p<.05. B) Image-J pixel density analysis of stem cell proteomic microarray. SOX2, TP63 and HCG are further increased in samples with increased TRA-1-60+ve cells. C) Inverted image of the scan of the array from TRA1-60+ve samples; D) Inverted image of the scan of the array from TRA1-60 –ve samples.</p

Disease status, age range and cytology results for cervical LBC samples obtained from the Scottish national HPV Archive.

<p>Disease status, age range and cytology results for cervical LBC samples obtained from the Scottish national HPV Archive.</p