A matter of new life and cell death: programmed cell death in the mammalian ovary.

BACKGROUND: The mammalian ovary is a unique organ that displays a distinctive feature of cyclic changes throughout the entire reproductive period. The estrous/menstrual cycles are associated with drastic functional and morphological rearrangements of ovarian tissue, including follicular development and degeneration, and the formation and subsequent atrophy of the corpus luteum. The flawless execution of these reiterative processes is impossible without the involvement of programmed cell death (PCD). MAIN TEXT: PCD is crucial for efficient and careful clearance of excessive, depleted, or obsolete ovarian structures for ovarian cycling. Moreover, PCD facilitates selection of high-quality oocytes and formation of the ovarian reserve during embryonic and juvenile development. Disruption of PCD regulation can heavily impact the ovarian functions and is associated with various pathologies, from a moderate decrease in fertility to severe hormonal disturbance, complete loss of reproductive function, and tumorigenesis. This comprehensive review aims to provide updated information on the role of PCD in various processes occurring in normal and pathologic ovaries. Three major events of PCD in the ovary-progenitor germ cell depletion, follicular atresia, and corpus luteum degradation-are described, alongside the detailed information on molecular regulation of these processes, highlighting the contribution of apoptosis, autophagy, necroptosis, and ferroptosis. Ultimately, the current knowledge of PCD aberrations associated with pathologies, such as polycystic ovarian syndrome, premature ovarian insufficiency, and tumors of ovarian origin, is outlined. CONCLUSION: PCD is an essential element in ovarian development, functions and pathologies. A thorough understanding of molecular mechanisms regulating PCD events is required for future advances in the diagnosis and management of various disorders of the ovary and the female reproductive system in general.

Optimized Transcriptional Signature for Evaluation of MEK/ERK Pathway Baseline Activity and Long-Term Modulations in Ovarian Cancer.

Ovarian cancer is the most aggressive and lethal of all gynecologic malignancies. The high activity of the MEK/ERK signaling pathway is tightly associated with tumor growth, high recurrence rate, and treatment resistance. Several transcriptional signatures were proposed recently for evaluation of MEK/ERK activity in tumor tissue. In the present study, we validated the performance of a robust multi-cancer MPAS 10-gene signature in various experimental models and publicly available sets of ovarian cancer samples. Expression of four MPAS genes (PHLDA1, DUSP4, EPHA2, and SPRY4) displayed reproducible responses to MEK/ERK activity modulations across several experimental models in vitro and in vivo. Levels of PHLDA1, DUSP4, and EPHA2 expression were also significantly associated with baseline levels of MEK/ERK pathway activity in multiple human ovarian cancer cell lines and ovarian cancer patient samples available from the TCGA database. Initial platinum therapy resistance and advanced age at diagnosis were independently associated with poor overall patient survival. Taken together, our results demonstrate that the performance of transcriptional signatures is significantly affected by tissue specificity and aspects of particular experimental models. We therefore propose that gene expression signatures derived from comprehensive multi-cancer studies should be always validated for each cancer type.

Drug toxicity assessment: cell proliferation versus cell death.

Analysis of the toxicity of chemotherapeutic drugs is one of the main tasks of clinical pharmacology. Decreased viability of tumor cells may reflect two important physiological processes, namely the arrest of proliferation associated with disturbances in cellular metabolism or actual cell death. Elucidation of the exact processes mediating a reduction in the number of cells is fundamentally important to establish the mechanisms of drug action. Only the use of a combination of cell biological and biochemical approaches makes it possible to understand these mechanisms. Here, using various lines of tumor cells and a set of methodological approaches, we carried out a detailed comparative analysis and demonstrated the possible ways to overcome the uncertainties in establishing the mechanisms of cell response to the action of chemotherapeutic drugs and their toxicity.

FOXM1-AKT Positive Regulation Loop Provides Venetoclax Resistance in AML.

Forkhead box protein M1 (FOXM1) is a crucial regulator of cancer development and chemoresistance. It is often overexpressed in acute myeloid leukemia (AML) and is associated with poor survival and reduced efficacy of cytarabine therapy. Molecular mechanisms underlying high FOXM1 expression levels in malignant cells are still unclear. Here we demonstrate that AKT and FOXM1 constitute a positive autoregulatory loop in AML cells that sustains high activity of both pro-oncogenic regulators. Inactivation of either AKT or FOXM1 signaling results in disruption of whole loop, coordinated suppression of FOXM1 or AKT, respectively, and similar transcriptomic changes. AML cells with inhibited AKT activity or stable FOXM1 knockdown display increase in HOXA genes expression and BCL2L1 suppression that are associated with prominent sensitization to treatment with Bcl-2 inhibitor venetoclax. Taken together, our data indicate that AKT and FOXM1 in AML cells should not be evaluated as single independent regulators but as two parts of a common FOXM1-AKT positive feedback circuit. We also report for the first time that FOXM1 inactivation can overcome AML venetoclax resistance. Thus, targeting FOXM1-AKT loop may open new possibilities in overcoming AML drug resistance and improving outcomes for AML patients.

Novel FOXM1 inhibitor identified via gene network analysis induces autophagic FOXM1 degradation to overcome chemoresistance of human cancer cells.

FOXM1 transcription factor is an oncogene and a master regulator of chemoresistance in multiple cancers. Pharmacological inhibition of FOXM1 is a promising approach but has proven to be challenging. We performed a network-centric transcriptomic analysis to identify a novel compound STL427944 that selectively suppresses FOXM1 by inducing the relocalization of nuclear FOXM1 protein to the cytoplasm and promoting its subsequent degradation by autophagosomes. Human cancer cells treated with STL427944 exhibit increased sensitivity to cytotoxic effects of conventional chemotherapeutic treatments (platinum-based agents, 5-fluorouracil, and taxanes). RNA-seq analysis of STL427944-induced gene expression changes revealed prominent suppression of gene signatures characteristic for FOXM1 and its downstream targets but no significant changes in other important regulatory pathways, thereby suggesting high selectivity of STL427944 toward the FOXM1 pathway. Collectively, the novel autophagy-dependent mode of FOXM1 suppression by STL427944 validates a unique pathway to overcome tumor chemoresistance and improve the efficacy of treatment with conventional cancer drugs.

The MEK1/2 Pathway as a Therapeutic Target in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is the deadliest of gynecological cancers due to its high recurrence rate and acquired chemoresistance. RAS/MEK/ERK pathway activation is linked to cell proliferation and therapeutic resistance, but the role of MEK1/2-ERK1/2 pathway in HGSOC is poorly investigated. We evaluated MEK1/2 pathway activity in clinical HGSOC samples and ovarian cancer cell lines using immunohistochemistry, immunoblotting, and RT-qPCR. HGSOC cell lines were used to assess immediate and lasting effects of MEK1/2 inhibition with trametinib in vitro. Trametinib effect on tumor growth in vivo was investigated using mouse xenografts. MEK1/2 pathway is hyperactivated in HGSOC and is further stimulated by cisplatin treatment. Trametinib treatment causes cell cycle arrest in G1/0-phase and reduces tumor growth rate in vivo but does not induce cell death or reduce fraction of CD133+ stem-like cells, while increasing expression of stemness-associated genes instead. Transient trametinib treatment causes long-term increase in a subpopulation of cells with high aldehyde dehydrogenase (ALDH)1 activity that can survive and grow in non-adherent conditions. We conclude that MEK1/2 inhibition may be a promising approach to suppress ovarian cancer growth as a maintenance therapy. Promotion of stem-like properties upon MEK1/2 inhibition suggests a possible mechanism of resistance, so a combination with CSC-targeting drugs should be considered.

Shift in VEGFA isoform balance towards more angiogenic variants is associated with tumor stage and differentiation of human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common and aggressive type of malignant liver tumor. HCC progression depends significantly on its vascularization and formation of new blood vessels. Vascular endothelial growth factor A (VEGFA) is a crucial regulator of tumor vascularization and components of VEGF-induced cell signaling pathways are important targets of therapeutical drugs that demonstrated the highest efficiency in case of advanced HCC (sorafenib and regorafenib). VEGFA is expressed as a set of isoforms with different functional properties, thus VEGFA isoform expression pattern may affect tumor sensitivity to anti-angiogenic drugs. However, information about VEGFA isoforms expression in HCC is still incomplete and contradictory. The present study aims to quantitatively investigate VEGFA isoform expression aberrations in HCC tissue. METHODS: A total of 50 pairs of HCC and non-tumor tissue samples were used to evaluate the VEGFA isoform spectrum using RT-PCR and quantitatively estimate changes in isoform expression using RT-qPCR. Correlations between these changes and tumor clinicopathological characteristics were analyzed. RESULTS: We identified VEGFA-189, VEGFA-165, and VEGFA-121 as predominant isoforms in liver tissue. Anti-angiogenic VEGFA-xxxb variants constituted no more than 5% of all mature VEGFA transcripts detected and their expression was not changed significantly in HCC tissue. We demonstrated for the first time that the least active variant VEGFA-189 is frequently repressed in HCC (p < 0.001), while no uniform changes were detected for potent angiogenesis stimulators VEGFA-165 and VEGFA-121. Isoform balance in HCC shifts from VEGFA-189 towards VEGFA-165 or VEGFA-121 in the majority of cases (p < 0.001). Changes in fractions, but not expression levels, of VEGFA-189 (decrease) and VEGFA-121 (increase) correlated with advanced Tumor-Node-Metastasis (TNM) and Barcelona Clinic Liver Cancer (BCLC) tumor stages (p < 0.05), VEGFA-189 fraction reduction was also associated with poor tumor differentiation (p < 0.05). DISCUSSION: A distinct shift in VEGFA isoform balance towards more pro-angiogenic variants occurs in HCC tissue and may modulate overall impact of VEGFA signaling. We suppose that the ratio between VEGFA isoforms is an important parameter governing HCC angiogenesis that may affect HCC progression and be used for optimizing the strategy of HCC therapy by predicting the response to anti-angiogenic drugs.

Effect of method of deduplication on estimation of differential gene expression using RNA-seq.

BACKGROUND: RNA-seq is a useful tool for analysis of gene expression. However, its robustness is greatly affected by a number of artifacts. One of them is the presence of duplicated reads. RESULTS: To infer the influence of different methods of removal of duplicated reads on estimation of gene expression in cancer genomics, we analyzed paired samples of hepatocellular carcinoma (HCC) and non-tumor liver tissue. Four protocols of data analysis were applied to each sample: processing without deduplication, deduplication using a method implemented in SAMtools, and deduplication based on one or two molecular indices (MI). We also analyzed the influence of sequencing layout (single read or paired end) and read length. We found that deduplication without MI greatly affects estimated expression values; this effect is the most pronounced for highly expressed genes. CONCLUSION: The use of unique molecular identifiers greatly improves accuracy of RNA-seq analysis, especially for highly expressed genes. We developed a set of scripts that enable handling of MI and their incorporation into RNA-seq analysis pipelines. Deduplication without MI affects results of differential gene expression analysis, producing a high proportion of false negative results. The absence of duplicate read removal is biased towards false positives. In those cases where using MI is not possible, we recommend using paired-end sequencing layout.