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.

New Born Calf Serum Can Induce Spheroid Formation in Breast Cancer KAIMRC1 Cell Line.

Three-dimensional (3D) cell culture systems have become very popular in the field of drug screening and discovery. There is an immense demand for highly efficient and easy methods to produce 3D spheroids in any cell format. We have developed a novel and easy method to produce spheroids from the newly isolated KAIMRC1 cell line in vitro. It can be used as a 3D model to study proliferation, differentiation, cell death, and drug response of cancer cells. Our procedure requires growth media supplemented with 10% new born calf serum (NBCS) and regular cell culture plates to generate KAIMRC1 spheroids without the need for any specialized 3D cell culture system. This procedure generates multiple spheroids within a 12-24-h culture. KAIMRC1 spheroids are compact, homogeneous in size and morphology with a mean size of 55.8 µm (±3.5). High content imaging (HCI) of KAIMRC1 spheroids treated with a panel of 240 compounds resulted in the identification of several highly specific compounds towards spheroids. Immunophenotyping of KAIMRC1 spheroids revealed phosphorylation of FAK, cJUN, and E-cadherin, which suggests the involvement of JNK/JUN pathway in the KAIMRC1 spheroids formation. Gene expression analysis showed upregulation of cell junction genes, GJB3, DSC1, CLDN5, CLDN8, and PLAU. Furthermore, co-culture of KAIMRC1 cells with primary cancer-associated-fibroblasts (CAFs) showcased the potential of these cells in drug discovery application.

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.

Preparation of iron oxide mesoporous magnetic microparticles as novel multidrug carriers for synergistic anticancer therapy and deep tumor penetration.

The preparation of mesoporous iron oxides with controllable physiochemical properties for effective therapeutic drug delivery remains a formidable challenge. Herein, iron oxide mesoporous magnetic microparticles (IO-MMMs) were prepared by a modified reverse hard-templating approach using, for the first time, acid-prepared mesoporous spheres (APMS) as the hard silica template. The obtained mesostructures exhibited remarkably high surface area and large pore volumes (SBET = 240 m2/g and Vpore = 0.55 cm3/g), controllable average sizes, generally uniform morphologies, and excellent biocompatibilities, allowing them to achieve optimal drug release in cancer cells and tumor tissues. IO-MMM carriers were able to co-load high amounts of hydrophilic chemotherapeutic drugs (Dox or Daun) and/or hydrophobic hormonal anticancer drugs (Tam), and release them sustainably in a pH-dependent manner, utilizing the fluorescence of Daun to real-time trace the intracellular drug distribution, and employing Daun/Tam to treat cancer by combined chemo/hormonal therapy. Cytotoxicity assays against different types of cancerous cells showed that the combinatory Daun/Tam@IO-MMM formulation significantly reduced the viability of metastatic MCF7 and KAIMRC1 breast as well as HCT8 colorectal cancer cells, with the least potency towards non-cancerous normal primary cells (up to 10-fold). Electron, flow, and live confocal microscopy imaging confirmed that the loaded vehicles were successfully and differentially uptaken by the different tested cells, gradually releasing their payloads, and causing apoptotic cell death. Importantly, compared to free drugs, Daun/Tam@IO-MMMs displayed enhanced drug accumulation in patient breast primary tumor tissues, deeply penetrating into the tumor region and killing the tumor cells inside. The designed carriers described here, thus, constitute a novel promising magnetic mesoporous smart system that entraps different kinds of drugs and release them in a controlled manner for combinatorial chemo/hormonal cancer theranostics. This multifactorial platform may open new avenues in cancer therapy as efficient synergistic antitumor system through overcoming limitations of conventional cancer therapy.

Isolation and characterization of a new naturally immortalized human breast carcinoma cell line, KAIMRC1.

BACKGROUND: Breast cancer is one of the most common cancer and a leading cause of death in women. Up to date the most commonly used breast cancer cell lines are originating from Caucasians or Afro-Americans but rarely cells are being derived from other ethnic groups. Here we describe for the first time the establishment of a naturally transformed breast cancer cell line, KAIMRC1 from an Arab woman of age 62 suffering from stage IIB breast cancer (T2N1M0). Moreover, we have characterized these cells for the biological and molecular markers, induction of MAPK pathways as well as its response to different commercially available drugs and compounds. METHODS: Breast cancer tissue sections were minced and cultured in media for several weeks. KAIMRC1 cells were successfully isolated from one of the primary breast tumor tissue cultures without any enzymatic digestion. To study the growth characteristics of the cells, wound healing assay, clonogenic assay, cell proliferation assays and live cell time-lapse microscopy was performed. Karyotyping, Immunophenotyping and molecular pathway specific compound treatment was also performed. A selective breast cancer gene expression panel was used to identify genes involved in the signal transduction dysregulation and malfunction of normal biological processes during breast carcinogenesis. RESULTS: These cells are ER/PR-positive and HER2-negative. The epithelial nature of these cells was confirmed by flow cytometry analysis using epithelial cell markers. They are cuboidal in shape and relatively smaller in size as compared to established cell lines, MCF-7, MDA MB-231 and the normal breast cell line, MCF-10A. In normal cell culture conditions these cells showed the capability of growing both in monolayer as well as in 3-D conformation. They showed a doubling time in vitro of approximately 24 h. They exhibit a modal karyotype of 58-63,X with abnormalities in a couple of chromosomes. KAIMRC1 cells were found to be more responsive to drug treatment in vitro in comparison to the established MDA MB-231 and MCF-7 cell lines. CONCLUSIONS: In conclusion we have isolated and characterized a new naturally immortalized breast cell line, KAIMRC1 with a potential to play a key role in opening up novel avenues towards the understanding of breast carcinoma.