Discovery of a novel potentially transforming somatic mutation in CSF2RB gene in breast cancer.

The colony stimulating factor 2 receptor subunit beta (CSF2RB) is the common signaling subunit of the cytokine receptors for IL-3, IL-5, and GM-CSF. Several studies have shown that spontaneous and random mutants of CSF2RB can lead to ligand independence in vitro. To date, no report(s) have been shown for the presence of potentially transforming and oncogenic CSF2RB mutation(s) clinically in cancer patients until the first reported case of a leukemia patient in 2016 harboring a germline-activating mutation (R461C). We combined exome sequencing, pathway analyses, and functional assays to identify novel somatic mutations in KAIMRC1 cells and breast tumor specimen. The patient's peripheral blood mononuclear cell (PBMC) exome served as a germline control in the identification of somatic mutations. Here, we report the discovery of a novel potentially transforming and oncogenic somatic mutation (S230I) in the CSF2RB gene of a breast cancer patient and the cell line, KAIMRC1 established from her breast tumor tissue. KAIMRC1 cells are immortalized and shown to survive and proliferate in ligand starvation condition. Immunoblot analysis showed that mutant CSF2RB signals through JAK2/STAT and PI3K/mTOR pathways in ligand starvation conditions. Screening a small molecule kinase inhibitor library revealed potent JAK2 inhibitors against KAIMRC1 cells. We, for the first time, identified a somatic, potentially transforming, and oncogenic CSF2RB mutation (S230I) in breast cancer patients that seem to be an actionable mutation leading to the development of new therapeutics for breast cancer.

Isolation and Establishment of a Highly Proliferative, Cancer Stem Cell-Like, and Naturally Immortalized Triple-Negative Breast Cancer Cell Line, KAIMRC2.

In vitro studies of a disease are key to any in vivo investigation in understanding the disease and developing new therapy regimens. Immortalized cancer cell lines are the best and easiest model for studying cancer in vitro. Here, we report the establishment of a naturally immortalized highly tumorigenic and triple-negative breast cancer cell line, KAIMRC2. This cell line is derived from a Saudi Arabian female breast cancer patient with invasive ductal carcinoma. Immunocytochemistry showed a significant ratio of the KAIMRC2 cells' expressing key breast epithelial and cancer stem cells (CSCs) markers, including CD47, CD133, CD49f, CD44, and ALDH-1A1. Gene and protein expression analysis showed overexpression of ABC transporter and AKT-PI3Kinase as well as JAK/STAT signaling pathways. In contrast, the absence of the tumor suppressor genes p53 and p73 may explain their high proliferative index. The mice model also confirmed the tumorigenic potential of the KAIMRC2 cell line, and drug tolerance studies revealed few very potent candidates. Our results confirmed an aggressive phenotype with metastatic potential and cancer stem cell-like characteristics of the KAIMR2 cell line. Furthermore, we have also presented potent small molecule inhibitors, especially Ryuvidine, that can be further developed, alone or in synergy with other potent inhibitors, to target multiple cancer-related pathways.

Proteomics Profiling of KAIMRC1 in Comparison to MDA-MB231 and MCF-7.

Proteomics characterization of KAIMRC1 cell line, a naturally immortalized breast cancer cells, is described in comparison to MCF-7 and MDA-MB-231 breast cancer cells. Quantitative proteomics analysis using the tandem mass tag (TMT)-labeled technique in conjunction with the phosphopeptide enrichment method was used to perform comparative profiling of proteins and phosphoproteins in the three cell lines. In total, 673 proteins and 33 Phosphoproteins were differentially expressed among these cell lines. These proteins are involved in several key cellular pathways that include DNA replication and repair, splicing machinery, amino acid metabolism, cellular energy, and estrogen signaling pathway. Many of the differentially expressed proteins are associated with different types of tumors including breast cancer. For validation, 4 highly significant expressed proteins including S-methyl-5'-thioadenosine phosphorylase (MTAP), BTB/POZ domain-containing protein (KCTD12), Poly (ADP-ribose) polymerase 1 (PARP 1), and Prelamin-A/C were subjected to western blotting, and the results were consistent with proteomics analysis. Unlike MCF-7 and MDA-MB-231, KAIMRC1 showed different phospho- and non-phosphoproteomic phenotypes which make it a potential model to study breast cancer.

Nuclear Receptors Are Differentially Expressed and Activated in KAIMRC1 Compared to MCF7 and MDA-MB231 Breast Cancer Cells.

We recently established a KAIMRC1 cell line that has unique features compared to the known breast cancer cell lines, MCF7 and MDA-MB231. To characterize it further, we investigated the expression profile of nuclear receptors and their respective co-factors in these cell lines. We confirm that in contrast to the triple negative cell line MDA-MB231, the MCF7 and KAIMRC1 are estrogen receptor alpha (ERa) and progesterone receptor alpha (PRa) positive, with significant lower expression of these receptors in KAIMRC1. KAIMRC1 cell is a vitamin D receptor (VDR) negative and V-ErbA-Related Protein 2 (EAR2) positive in contrast to MCF7 and MDA-MB231. Remarkably, the histone deacetylases (HDACs) are highly expressed in KAIRMC1 with HDAC6 and HDAC 7 are exclusively expressed in KAIMRC1 while thyroid hormone receptor-associated protein 80 (TRAP80), telomeric DNA binding protein 1 (TBP1) and TGF-beta receptor interacting protein (TRIP1) are absent in KAIMRC1 but present in MCF7 and MDA-MB231. In a luciferase reporter assay, the ERa coexpression is needed for estrogen receptor element (ERE)-luciferase activation by estradiol in KAIMRC1 but not in MCF7. The co-expression of exogenous Liver X receptor alpha (LXRa)/retinoid X receptor alpha (RXRa) are necessary for LXR responsive element (LXRE) activation by the GW3696 in the three cell lines. However, the activity of peroxisome proliferator-activated receptor response element (PPARE)-tk-luciferase reporter increased when peroxisome proliferator-activated receptors alpha (PPARa)/RXRa were coexpressed but the addition of PPARa agonist (GW7647) did not stimulate further the reporter. The signal of the PPARE reporter increased in a dose-dependent manner with rosiglitazone (PPARg agonist) in KAIMRC1, MCF7, and MDA-MB231 when the proliferator-activated receptors gamma (PPARg)/RXRa receptors were cotransfected. Retinoic acid-induced activation of retinoic acid receptor response element (RARE)-tk-luciferase is dependent on exogenous expression of retinoic acid receptor alpha (RARa)/RXRa heterodimer in MDA-MB 231 but not in MCF7 and KAIMRC1 cell lines. In the three cell lines, Bexarotene-induced retinoid X receptor response element (RXRE)-luciferase reporter activation was induced only if the RXRa/LXRa heterodimer were co-expressed. The vitamin D receptor response element (VDRE)-luciferase reporter activity showed another distinct feature of KAIMRC1, where only co-expression of exogenous vitamin D receptor (VDR)/RXRa heterodimer was sufficient to reach the maximum rate of activation of VDRE reporter. In the proliferation assay, nuclear receptors ligands showed a distinct effect on KAIMRC1 compared to MCF7 and MDA-MB231. Growth inhibition effects of used ligands suggest that KAIMRC1 correlate more closely to MDA-MB231 than MCF7. Vitamin D3, rosiglitazone, novel RXR compound (RXRc) and PPARa compound (GW6471) have the most profound effects. In conclusion, we showed that nuclear receptors are differentially expressed, activated and also their ligand produced distinct effects in KAIMRC1 compared to MCF7 and MDA-MB231. This finding gives us confidence that KAIMRC1 has a unique biological phenotype.

Identification of carbapenem-resistant Pseudomonas aeruginosa in selected hospitals of the Gulf Cooperation Council States: dominance of high-risk clones in the region.

PURPOSE: The molecular epidemiology and resistance mechanisms of carbapenem-resistant Pseudomonas aeruginosa (CRPA) were determined in hospitals in the countries of the Gulf Cooperation Council (GCC), namely, Saudi Arabia, the United Arab Emirates, Oman, Qatar, Bahrain and Kuwait. METHODOLOGY: Isolates were screened for common carbapenem-resistance genes by PCR. Relatedness between isolates was assessed using previously described genotyping methods: an informative-single nucleotide polymorphism MassARRAY iPLEX assay (iPLEX20SNP) and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay, with selected isolates being subjected to multilocus sequence typing (MLST). Ninety-five non-repetitive isolates that were found to be resistant to carbapenems were subjected to further investigation.Results/Key findings. The most prevalent carbapenemase-encoding gene, blaVIM-type, was found in 37/95 (39 %) isolates, while only 1 isolate (from UAE) was found to have blaIMP-type. None of the CRPA were found to have blaNDM-type or blaKPC-type. We found a total of 14 sequence type (ST) clusters, with 4 of these clusters being observed in more than 1 country. Several clusters belonged to the previously recognized internationally disseminated high-risk clones ST357, ST235, ST111, ST233 and ST654. We also found the less predominant ST316, ST308 and ST823 clones, and novel MLST types (ST2010, ST2011, ST2012 and ST2013), in our collection. CONCLUSION: Overall our data show that 'high-risk' CRPA clones are now detected in the region and highlight the need for strategies to limit further spread of such organisms, including enhanced surveillance, infection control precautions and further promotion of antibiotic stewardship programmes.