Loss of Mediator complex subunit 13 (MED13) promotes resistance to alkylation through cyclin D1 upregulation.

Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR-Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified cyclin D1 (CCND1) as one of the highly overexpressed genes in MED13 knock-out (KO) cells, characterized by shorter G1 phase. MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.

Potential Antiviral Options against SARS-CoV-2 Infection.

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.

Genome-wide analysis of the oxyntic proliferative isthmus zone reveals ASPM as a possible gastric stem/progenitor cell marker over-expressed in cancer.

The oxyntic proliferative isthmus zone contains the main stem/progenitor cells that provide for physiological renewal of the distinct mature cell lineages in the oxyntic epithelium of the stomach. These cells are also proposed to be the potential cells-of-origin of gastric cancer, although little is known about their molecular characteristics and specific biological markers are lacking. In this study, we developed a method for serial section-navigated laser microdissection to isolate cells from the proliferative isthmus zone of rat gastric oxyntic mucosa for genome-wide microarray gene expression analysis. Enrichment analysis showed a distinct gene expression profile for the isthmus zone, with genes regulating intracellular processes such as the cell cycle and ribosomal activity. The profile was also related to stem cell transcriptional networks and stomach neoplasia. Genes expressed uniquely in the isthmus zone were associated with E2F transcription factor 1 (E2F1), which participates in the self-renewal of stem cells and in gastric carcinogenesis. One of the unique genes was Aspm [Asp (abnormal spindle) homologue, microcephaly-associated (Drosophila)]. Here we show ASPM in single scattered epithelial cells located in the proliferative isthmus zone of rat, mouse and human oxyntic mucosa, which do not seem to be actively dividing. The ASPM-expressing cells are mainly mature cell marker-deficient, except for a limited overlap with cells with neuroendocrine and tuft cell features. Further, both ASPM and E2F1 were expressed in human gastric cancer cell lines and increased and correlated in human gastric adenocarcinomas compared to non-tumour mucosa, as shown by expression profile analyses and immunohistochemistry. The association between ASPM and the transcription factor E2F1 in gastric tissue is relevant, due to their common involvement in crucial cell fate-regulatory mechanisms. Our results thus introduce ASPM as a novel possible oxyntic stem/progenitor cell marker that may be involved in both normal gastric physiology and gastric carcinogenesis.

Molecular resistance fingerprint of pemetrexed and platinum in a long-term survivor of mesothelioma.

BACKGROUND: Pemetrexed, a multi-folate inhibitor combined with a platinum compound is the first-line treatment of malignant mesothelioma, but median survival is still one year. Intrinsic and acquired resistance to pemetrexed is common, but its biological basis is obscure. Here we report for the first time a genome-wide profile of acquired resistance in the tumour from an exceptional case with advanced pleural mesothelioma and almost six years survival after 39 cycles of second-line pemetrexed/carboplatin treatment. METHODOLOGY AND PRINCIPAL FINDINGS: Genome-wide analysis with Illumina BeadChip Kit of 25,000 genes was performed on mRNA from pre-treatment and post-resistance biopsies from this individual as well on case and control samples from our previously published study (in total 17 samples). Cell specific expression of proteins encoded by selected genes were analysed by immunohistochemistry. Serial serum levels of CA125, CYFRA21-1 and SMRP levels were examined. TS protein, the main target of pemetrexed was overexpressed. Proteins and genes related to DNA damage response, elongation and telomere extension and repair related directly and indirectly to platinum resistance were overexpressed, as the CHK1 protein and the genes CHEK2, LIG3, POLD1, POLA2, FANCD2, PRPF19, RECQ5 respectively, the last two not previously described in mesothelioma. We observed a down-regulation of leukocyte transendothelial migration and cell adhesion molecules pathways. Silencing of NT5C in two mesothelioma cell lines did not sensitize the cells to Pemetrexed. Proposed resistance markers are TS, KRT7/ CK7, TYMP/ thymidine phosphorylase and down-regulated SPARCL1 and CDKN1B. Moreover, comparison of the primary expression of the sensitive versus a primary resistant case showed multi-fold overexpressed DNA repair, cell cycle, cytokinesis, and spindle formation in the latter. Serum CA125 and SMRP reflected the clinical and radiological course and tumour burden. CONCLUSIONS: Genome-wide microarray of mesothelioma pre- and post-resistance biopsies indicated a novel resistance signature to pemetrexed/carboplatin that deserve validation in a larger cohort.

Gastrin upregulates the prosurvival factor secretory clusterin in adenocarcinoma cells and in oxyntic mucosa of hypergastrinemic rats.

We show that the gastric hormone gastrin induces the expression of the prosurvival secretory clusterin (sCLU) in rat adenocarcinoma cells. Clusterin mRNA was still upregulated in the presence of the protein synthesis inhibitor cycloheximide, although at a lower level. This indicates that gastrin induces clusterin transcription independently of de novo protein synthesis but requires de novo protein synthesis of signal transduction pathway components to achieve maximal expression level. Luciferase reporter assay indicates that the AP-1 transcription factor complex is involved in gastrin-mediated activation of the clusterin promoter. Gastrin-induced clusterin expression and subsequent secretion is dependent on sustained treatment, because removal of gastrin after 1-2 h abolished the response. Neutralization of secreted clusterin by a specific antibody abolished the antiapoptotic effect of gastrin on serum starvation-induced apoptosis, suggesting that extracellular clusterin is involved in gastrin-mediated inhibition of apoptosis. The clusterin response to gastrin was validated in vivo in hypergastrinemic rats, showing increased clusterin expression in the oxyntic mucosa, as well as higher levels of clusterin in plasma. In normal rat oxyntic mucosa, clusterin protein was strongly expressed in chromogranin A-immunoreactive neuroendocrine cells, of which the main cell type was the histidine decarboxylase-immunoreactive enterochromaffin-like (ECL) cell. The association of clusterin with neuroendocrine differentiation was further confirmed in human gastric ECL carcinoids. Interestingly, in hypergastrinemic rats, clusterin-immunoreactive cells formed distinct groups of diverse cells at the base of many glands. Our results suggest that clusterin may contribute to gastrin's growth-promoting effect on the oxyntic mucosa.

Octreotide induces apoptosis in the oxyntic mucosa.

Previous studies show that octreotide LAR causes regression of gastric ECL-cell carcinoids, reducing both number and size of tumours. This study examines the molecular mechanisms behind the antiproliferative effect of octreotide on the oxyntic mucosa. Female rats received octreotide LAR for 21 days. Serum gastrin was measured and tissue samples for RNA extraction and histology collected from the oxyntic mucosa. Affymetrix analysis showed regulated genes related to apoptosis and proliferation, and a large group of regulated growth-related transcription factors. Verification by real time qRT-PCR showed a high degree of consistency to the microarray results. Supporting the molecular results, histomorphometry showed significant decreases in the number of gastric glands, cells per gland and length of glands, and a tendency towards increased apoptosis and decreased proliferation. Thus, octreotide exerts a negative effect on oxyntic mucosal growth, and induces extensive gene expression changes relevant to growth regulation.

Gene expression based classification of gastric carcinoma.

The aim of the present work is to identify molecular markers that allow classification of gastric carcinoma with respect to important clinicopathological parameters. Gastric adenocarcinomas were subjected to cDNA microarray analysis with a 2.504 gene probe set. Using the Rosetta rough-set based learning system, good classifiers were generated for gene-expression based prediction of intestinal or diffuse growth pattern according to Laurén's classification and presence of lymph node metastases. To our knowledge, this is the first study on gastric carcinoma in which molecular classification has been achieved for more than one clinicopathological parameter based on microarray gene expression profiles.