Caspases as Putative Biomarkers of Cervical Cancer Development

Resistance to apoptosis is commonly accepted as the principal hallmark of a cancer cell, while caspases are recognized as the key molecular players of the apoptosis regulatory network. Since the level of caspase activity is thought to be directly coupled with aggressive features of cancer cells (such as ability to withstand immune reactions, invasiveness, drug resistance, etc.), these proteases could serve as objective diagnostic markers especially for those types of cancer where early differential diagnosis is needed. Cervical cancer develops through morphologically well-described stages—from intraepithelial lesions of 1/2/3 grade including carcinoma in situ to microinvasive and invasive cancer with precancerous lesions known to be potentially reversible. The percentage of cervical neoplasms diagnosed at early stages is relatively high, providing a basis for the use of cervical cancer as an in vivo model to investigate the mechanisms of apoptosis modulation in malignant cells. The existing diagnostic criteria, despite their usefulness, have substantial limitations with respect to cervical cancer and preneoplastic lesions, so caspases may be helpful in improving them, but there is insufficient data regarding the involvement of these enzymes in cervical cancer development. In this chapter, we report on specific patterns of activity of caspases revealed in tissue biopsies and blood lymphocytes in association with different stages of cervical cancer development. The data indicate that caspases are pivotal components of the in vivo molecular “portrait” of cervical cancer and have the potential of being used as biomarkers

Characteristics of immune-active and immune-silent phenotypes of early-stage cervical carcinoma as revealed by transcriptome sequencing

Molecular classification, immuneheterogeneity, and the existence of distinct immunophenotypes of virus-associated cervical cancer (CeCa) remain as-yet weakly explored issues, and this is particularly true of its earliest clinical stages and pre-invasive forms: cervical intraepithelial neoplastic (CIN) lesions. The goal of the study was to identify transcriptomic landscapes of invasive CeCa at its initial progression that differ substantially in their immune-related characteristics, patterns of signaling pathways and composition of the microenvironment. Transcriptome profiling was carried out using RNA-sequencing on Illumina platform. A panel of surgical-derived tissue samples comprised human papillomavirus-positive CIN grade 1-3, cancer of FIGO IA1-IIB stages, and morphologically normal epithelium. Transcriptomic profiles were analyzed with the use of bioinformatics tools, such as gene set enrichment (GAGE) for signaling pathways, xCell enrichment for cell composition identification, and PREDA positional analysis of genomic data. Hierarchical clustering revealed heterogeneity of transcriptomic profiles within the early-stage CeCa, namely, the existence of two clusters of tumor samples and three functional patterns of genes showing coordinately altered expression. Pathway enrichment analysis on genes differently expressed between the two clusters/groups of CeCa samples (‘A' and ‘B') and CIN (group ‘C') suggested that invasive tumor progression in groups ‘A' and ‘B' might rely on immunologically dissimilar mechanisms. xCell analysis confirmed heterogeneity of changes in the abundancies of cell populations when comparing CeCa sample groups and CIN, along with differences in immune and stromal scores. PREDA demonstrated that these transcriptomic differences could be linked to different chromosomal regions and co-localized with particular gene families and potentially the reported virus integration hotspots. Overall, the existence and detectability of different transcriptomic immune-based phenotypes of invasive CeCa at its initial stages of progression is shown, which may provide new options to broaden the knowledge and applicability of target and immune anti-cancer therapy

ANALYSIS OF APOPTOSIS FACTORS IN PATIENTS WITH PREINVASIVE AND INVASIVE CERVICAL CANCER

This article assesses the disturbance degree of apoptotic program in patients with preinvasive and invasive cervical cancer by investigating the expression level of genes of caspases-3, -6, -8 и -9 in mononuclear cells of periphery blood and in tumor tissue on the two regulating levels – on mRNA level (transcriptional) and proteolytic activity (post transcriptional). 75 patients with stage III of cervical intraepithelial neoplasias (CIN) III  (middle age of 32,9 ± 7,4),45 patients with stage IA (31,3 ± 6,0), 21 – with stage II (43,6 ± 13,2), 15 – with stage III–IV (46,9 ± 11,1) have been examined. The control group has been formed from 30 almost healthy donors without any cervical pathology and papilloma human virus (control 1) and 30 patients with a preinvasive and microinvasive cervical cancer (control 2). It has been found that in proportion of progress of cervix cancer, the membranous expression of CD95 increases in MPB – fraction (peripheral blood monocytes) when the a CIN and initial stages of cervix cancer, more than two times. Herewith number CD95+-lymphocytes is positively correlated with stage of cervical cancer (r = 0,91; R2  = 0,82; p << 0,01). It has been found out that the activity gain of caspase-8 (r = 0,92; R2  = 0,86; p << 0,01), caspase-6 (r = 0,77; R2 = 0,59; p << 0,01) and reduction activity of caspase-9 (r = –0,60;  R2 = 0,36; p < 0,01) in mononuclear cells of peripheral blood pointed out on the sensitivity increase to Fas-induced apoptosis. Opposite, in tumor tissue, beginning from CIN stage III, apoptosis-resistant phenotype is formed, it were defined by the expression of caspase-3 (r = –0,72; R2 = 0,52, p < 0,01), caspase-6 (r = –0,59; R2 = 0, 38; p < 0,01) и caspase-9 (r = –0,67; R2 = 0,45; p < 0,01) by mRNA level and proteolytic activity. It has been shown, that the cervical cancer development is accompanied by multilateral disturbances of apoptotic processes, which are realized in decreased function of caspases and multilateral resistance of tumor cells in against signals of apoptosis. The obtained results give an opportunity to examine the genes of caspase as a probable prospective biomarkers in complex diagnostic of a CIN and early diagnosis of a cervical cancer (in combination with morphological criteria and other molecular markers of viral origin

Sequencing-based transcriptome analysis reveals diversification of immune response- and angiogenesis-related expression patterns of early-stage cervical carcinoma as compared with high-grade CIN

BackgroundMolecular diversity of virus-associated cervical cancer remains a relatively underexplored issue, and interrelations of immunologic and angiogenic features during the establishment of a particular landscape of the cervical cancer microenvironment are not well-characterized, especially for its earliest clinical stages, although this may provide insight into the mechanisms behind the differences in tumor aggressiveness, treatment responsiveness and prognosis. In this research, we were aimed at identifying transcriptomic landscapes of early-stage cervical carcinoma that differ substantially in their immune-related characteristics, patterns of signaling pathways and composition of the microenvironment in comparison with immediate precursor (intraepithelial) lesions.MethodsWe performed the Illumina platform-based RNA sequencing using a panel of fresh tissue samples that included human papillomavirus-positive cervical intraepithelial neoplastic lesions (CIN), invasive squamous carcinoma of the cervix of FIGO IA1-IIB stages, and morphologically normal epithelium. The derived transcriptomic profiles were bioinformatically analyzed and compared by patterns of signaling pathway activation, distribution of tumor-infiltrating cell populations, and genomic regions involved.ResultAccording to hierarchical cluster analysis of the whole-transcriptome profiles, tissue samples were distributed between three groups, or gene expression patterns (the one comprising most pre-cancer cases and the other two encompassing mostly early-stage invasive cancer cases). Differentially expressed genes were retrieved in each intergroup pairwise comparison followed by Gene Ontology analysis. Gene set enrichment analysis of the two groups of tumor samples in comparison with the CIN group identified substantial differences in immunological and angiogenic properties between tumorous groups suggesting the development of different molecular phenotypes. Cell composition analysis confirmed the diverse changes in the abundancies of immune and non-immune populations and, accordingly, different impacts of the immune and stromal compartments on the tumor microenvironment in these two groups of tumors compared to CIN. Positional gene expression analysis demonstrated that the identified transcriptomic differences were linked to different chromosomal regions and co-localized with particular gene families implicated in immune regulation, inflammation, cell differentiation, and tumor invasion.ConclusionsOverall, detection of different transcriptomic patterns of invasive cervical carcinoma at its earliest stages supports the diverse impacts of immune response- and angiogenesis-related mechanisms on the onset of tumor invasion and progression. This may provide new options for broadening the applicability and increasing the efficiency of target anti-angiogenic and immune-based therapy of virus-associated cervical carcinoma

T- and NK-cell populations with regulatory phenotype and markers of apoptosis in circulating lymphocytes of patients with CIN3 or microcarcinoma of the cervix: evidence for potential mechanisms of immune suppression

Abstract Background Processes and mechanisms responsible for systemic immune suppression in early-stage cervical cancer remain substantially underinvestigated. In this work, we focused on studying the frequencies of circulating regulatory T (CD4 and CD8 Tregs) and NK (NKregs) cells in parallel with assessment of apoptotic markers expression in T cells from patients with preinvasive and microinvasive cervical cancer, with the aim to determine whether up-regulation of apoptosis-associated markers in Т lymphocytes accompanies cervical cancer development and correlates with the change in percentages of regulatory cell populations at systemic level during the initial stages of invasive cervical cancer progression. Methods Fourty two women with histologically confirmed cervical intraepithelial neoplasia grade 3 (CIN3, including carcinoma in situ) or cervical cancer (stage IA) and 30 healthy women (control) were enrolled in the study. Peripheral blood samples were taken immediately before surgery or any treatment and immediately subjected to multicolor flow cytometry. Results Analysis of a combination of CD4/CD8, CD25, CD127, and FoxP3 markers revealed a statistically significant increase in the frequencies of Tregs within both the CD4 and CD8 subsets of circulating lymphocytes in patients with CIN3 and stage IA cancer. In contrast, lower numbers of NKregs (defined as CD16dim/negCD56bright subpopulation) and increased CD56dim/CD56bright NK ratio were found in patients compared to controls, with the percentage of CD16brightCD56dim cells (major subtype of circulating NKs) showing no difference. Patients also exhibited an increased expression of CD95 in total peripheral blood T lymphocytes, along with increased level of Annexin V binding to CD95-positive cells, suggesting higher susceptibility of T cells to apoptosis and potential involvement of CD95-dependent pathway in early-stage cervical cancer. Differential analysis of CD4 and CD8 T cells revealed different trends in the change of CD95 expression, confirming that this change likely has different functional significance for these two subsets. A search for correlations between the phenotypic parameters analyzed in this study was performed to demonstrate that women with early neoplastic lesions of the cervix, such as carcinoma in situ and microinvasive carcinoma, displayed a coordinated increase in expression of Treg markers in circulating lymphocytes, along with more pronounced cross-relationships between Treg numbers, CD95 expression on T cells, and apoptosis, compared to the control group. Conclusions The results of this study suggest that a diversity of immune regulatory mechanisms that provide support for initial stages of invasive growth in cervical cancer patients includes systemic changes in the ratios between the principal regulatory and effector lymphocyte populations both within adaptive and innate immunity

Early response genes in the pathogenesis of cancer of the cervix uteri: a review

Early response genes are a group of proto-oncogenes that are the first to be activated in cell stimulation with different growth factors and to be involved in the regulation of cell proliferation and differentiation. Large amount of information supporting that altered expression of these genes is one of the central and earliest events of carcinogenesis has been accumulated. In this connection, it is promising to use early response genes as diagnostic and prognostic markers for the detection and combination therapy of cancer of the cervix uteri, one of the most common gynecological malignancies characterized by high mortality rates and difficulties in early diagnosis. The theoretical basis for these promises is the found mechanisms for the interaction of early response genes with human papillomavirus genome, the main cause of cervix uteri cancer

The body’s immune response in the induction and progression of cancer of the cervix uteri: possible mechanisms

Human papillomavirus (HPV) that is a main cause of cancer of the cervix uteri (CCU) has immunogenic properties, i.e. an abilityto activate antiviral immunity responses as adaptive HPV-specific and innate ones. For this reason, despite multiple mechanisms generated by HPV to avoid immunity responses, the human body can eliminate the infection in most cases. At the same time, CCU results from the combined influence of many factors of different nature, among which the factors that impair the normal course of an immune response are of vital importance.This review describes the major factors and mechanisms, which promote the establishment of persistent HPV infection and the progression of dysplasia to cancer, on the one hand, and allow the tumor cells in CCU to restrict the body’s immune reactions, on the other Immune disorders induced by the virus and/or tumor cells are considered at both local and systemic levels. Particular emphasis is placed on the molecular mechanisms that can change the population composition and functional activity of leukocytes and the cytokine profile of cells and can form the tumor suppressor microenvironment