Background
The mutational status of ovarian cancer cell line IGROV-1 is inconsistent across the literature, suggestive of multiple clonal populations of the cell line. IGROV-1 has previously been ...categorised as an inappropriate model for high-grade serous ovarian cancer.
Methods
IGROV-1 cells were obtained from the Netherlands Cancer Institute (IGROV-1-NKI) and the MD Anderson Cancer Centre (IGROV-1-MDA). Cell lines were STR fingerprinted and had their chromosomal copy number analysed and BRCA1/2 genes sequenced. Mutation status of ovarian cancer-related genes were extracted from the literature.
Results
The IGROV-1-NKI cell line has a tetraploid chromosomal profile. In contrast, the IGROV-1-MDA cell line has pseudo-normal chromosomes. The IGROV-1-NKI and IGROV-MDA are both STR matches (80.7% and 84.6%) to the original IGROV-1 cells isolated in 1985. However, IGROV-1-NKI and IGROV-1-MDA are not an STR match to each other (78.1%) indicating genetic drift. The
BRCA1
and
BRCA2
gene sequences are 100% identical between IGROV-1-MDA and IGROV-1-NKI, including a
BRCA1
heterozygous deleterious mutation. The IGROV-1-MDA cells are more resistant to cisplatin and olaparib than IGROV-1-NKI. IGROV-1 has a mutational profile consistent with both Type I (
PTEN, PIK3CA and ARID1A
) and Type II ovarian cancer (
BRCA1, TP53
) and is likely to be a Type II high-grade serous carcinoma of the SET (Solid, pseudo-Endometroid and Transitional cell carcinoma-like morphology) subtype.
Conclusions
Routine testing of chromosomal copy number as well as the mutational status of ovarian cancer related genes should become the new standard alongside STR fingerprinting to ensure that ovarian cancer cell lines are appropriate models.
To prevent the development of endocrine-resistant breast cancer, additional targeted therapies are increasingly being trialled in combination with endocrine therapy. The molecular mechanisms ...facilitating cancer cell survival during endocrine treatment remain unknown but could help direct selection of additional targeted therapies. We present a novel proteomic timecourse dataset, profiling potential drug targets in a population of MCF7 cells during 1 year of tamoxifen treatment. Reverse phase protein arrays profiled >70 proteins across 30 timepoints. A biphasic response to tamoxifen was evident, which coincided with changes in growth rate. Tamoxifen strongly impeded cell growth for the first 160 days, followed by gradual growth recovery and eventual resistance development. The growth-impeded phase was distinguished by the phosphorylation of Stat3 (y705) and Src (y527). Tumour tissue from patients treated with neo-adjuvant endocrine therapy (<4 months) also displayed increased Stat3 and Src signalling. Inhibitors of Stat3 (napabucasin) and Src (dasatinib), were effective at killing tamoxifen-treated MCF7 and T47D cells. Sensitivity to both drugs was significantly enhanced once tamoxifen had induced the growth-impeded phase. This novel proteomic resource identifies key mechanisms enabling cell survival during tamoxifen treatment. It provides valuable insight into potential drug combinations and timing that may prevent the development of endocrine resistance.
Aberrant PI3K signalling is implicated in trastuzumab resistance in HER2-positive gastric cancer (GC). The role of PI3K or MEK inhibitors in sensitising HER2-positive GCs to trastuzumab or in ...overcoming trastuzumab resistance is unclear.
Using mass spectrometry-based genotyping we analysed 105 hotspot, non-synonymous somatic mutations in PIK3CA and ERBB-family (EGFR, ERBB2, ERBB3 and ERBB4) genes in gastric tumour samples from 69 patients. A panel of gastric cell lines (N87, OE19, ESO26, SNU16, KATOIII) were profiled for anti-proliferative response to the PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib alone and in combination with anti-HER2 therapies.
Patients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months). Mutations in PIK3CA were only identified in HER2-negative tumours, while ERBB-family mutations were identified in HER2-positive and HER2-negative tumours. Copanlisib had anti-proliferative effects in 4/5 cell lines, with IC50s ranging from 23.4 (N87) to 93.8 nM (SNU16). All HER2-positive cell lines except SNU16 were sensitive to lapatinib (IC50s 0.04 µM-1.5 µM). OE19 cells were resistant to trastuzumab. The combination of lapatinib and copanlisib was synergistic in ESO-26 and OE-19 cells (ED50: 0.83 ± 0.19 and 0.88 ± 0.13, respectively) and additive in NCI-N87 cells (ED50:1.01 ± 0.55). The combination of copanlisib and trastuzumab significantly improved growth inhibition compared to either therapy alone in NCI-N87, ESO26 and OE19 cells (p < 0.05).
PI3K or MEK inhibition alone or in combination with anti-HER2 therapy may represent an improved treatment strategy for some patients with HER2-positive GC, and warrants further investigation in a clinical trial setting.
We tested the hypothesis that inhibitor of apoptosis family (IAP) proteins may be altered in BRCA1-mutated ovarian cancers and that could affect the sensitivity to IAP inhibitors.
The levels of IAP ...proteins were evaluated in human cancers and cell lines. Cell lines were used to determine the effects of IAP inhibitors. The in vivo effects of treatments were evaluated in PDX mouse models.
Expression of X-linked inhibitor of apoptosis (XIAP) is increased in BRCA1-mutated cancers and high levels are associated with improved patient outcomes after platinum chemotherapy. XIAP overexpression is mediated by NF-kB activation and is associated with an optimisation of PARP. BRCA1-mutated cell lines are particularly sensitive to IAP inhibitors due to an inhibitory effect on PARP. Both a BRCA1-mutated cell line with acquired resistance to PARP inhibitors and one with restored BRCA1 remain sensitive to IAP inhibitors. Treatment with IAP inhibitors restores the efficacy of PARP inhibition in these cell lines. The IAP inhibitor LCL161 alone and in combination with a PARP inhibitor, exhibited antitumour effects in PDX mouse models of resistant BRCA2 and 1-mutated ovarian cancer, respectively.
A clinical trial may be justified to further investigate the utility of IAP inhibitors.
Participatory development literature involving community-based ecotourism management (CBEM) has only recently addressed issues pertaining to indigenous governance and decision-making systems. This ...paper contributes to sustainable tourism by presenting local decision-making practices and issues arising from the perspective of the members of one village in the Boumā National Heritage Park, Fiji. It shows that introduced democratic decision-making systems may not contribute to political empowerment in CBEM and can cause difficult situations. It is argued that greater attention to local systems of governance is required if tourism practitioners are to fully understand decision-making and participation in CBEM. The paper also offers a culturally appropriate methodology that may produce more meaningful outcomes for sustainable tourism research in indigenous Fijian contexts, and in other contexts worldwide. It argues that levels of empowerment should not just be treated as the outcome but as a part of the process of tourism development. It explores the core Fijian cultural concept of vanua as a way of life, involving interrelated social, ecological and spiritual elements. An emic perspective utilising informal talanoa (discussions) is used and examined, along with the roles of kin groups, village spokesmen and clan systems, and their relationship with western business decision-making practices.
Studies have demonstrated the efficacy of Palbociclib (CDK 4/6 inhibitor), Gedatolisib (PI3K/mTOR dual inhibitor) and PD0325901 (MEK1/2 inhibitor) in colorectal cancer (CRC), however single agent ...therapeutics are often limited by the development of resistance.
We compared the anti-proliferative effects of the combination of Gedatolisib and Palbociclib and Gedatolisib and PD0325901 in five CRC cell lines with varying mutational background and tested their combinations on total and phosphoprotein levels of signaling pathway proteins.
The combination of Palbociclib and Gedatolisib was superior to the combination of Palbociclib and PD0325901. The combination of Palbociclib and Gedatolisib had synergistic anti-proliferative effects in all cell lines tested CI range: 0.11-0.69 and resulted in the suppression of S6rp (S240/244), without AKT reactivation. The combination of Palbociclib and Gedatolisib increased BAX and Bcl−2 levels in PIK3CA mutated cell lines. The combination of Palbociclib and Gedatolisib caused MAPK/ERK reactivation, as seen by an increase in expression of total EGFR, regardless of the mutational status of the cells.
This study shows that the combination of Palbociclib and Gedatolisib has synergistic anti-proliferative effects in both wild-type and mutated CRC cell lines. Separately, the phosphorylation of S6rp may be a promising biomarker of responsiveness to this combination.
Mutations in BRCA1/2 increase the risk of developing breast and ovarian cancer. Germline BRCA1/2 mutations occur in 8.6–13.7% of unselected epithelial ovarian cancers, somatic mutations are also ...frequent. BRCA1/2 mutated or dysfunctional cells may be sensitive to PARP inhibition by synthetic lethality. The aim of this study is to comprehensively characterise the BRCA1/2 status of a large panel of ovarian cancer cell lines available to the research community to assist in biomarker studies of novel drugs and in particular of PARP inhibitors.
The BRCA1/2 genes were sequenced in 41 ovarian cell lines, mRNA expression of BRCA1/2 and gene methylation status of BRCA1 was also examined. The cytotoxicity of PARP inhibitors olaparib and veliparib was examined in 20 cell lines.
The cell line SNU-251 has a deleterious BRCA1 mutation at 5564G > A, and is the only deleterious BRCA1/2 mutant in the panel. Two cell lines (UPN-251 and PEO1) had deleterious mutations as well as additional reversion mutations that restored the protein functionality. Heterozygous mutations in BRCA1/2 were relatively common, found in 14.6% of cell lines. BRCA1 was methylated in two cell lines (OVCAR8, A1847) and there was a corresponding decrease in gene expression. The BRCA1 methylated cell lines were more sensitive to PARP inhibition than wild-type cells. The SNU-251 deleterious mutant was more sensitive to PARP inhibition, but only in a long-term exposure to correct for its slow growth rate. Cell lines derived from metastatic disease are significantly more resistant to veliparib (2.0 fold p = 0.03) compared to those derived from primary tumours. Resistance to olaparib and veliparib was correlated Pearsons-R 0.5393, p = 0.0311.
The incidence of BRCA1/2 deleterious mutations 1/41 cell lines derived from 33 different patients (3.0%) is much lower than the population incidence. The reversion mutations and high frequency of heterozygous mutations suggest that there is a selective pressure against BRCA1/2 in cell culture similar to the selective pressure seen in the clinic after treatment with chemotherapy. PARP inhibitors may be useful in patients with BRCA1 deleterious mutations or gene methylation.
► First study of BRCA1/2 mutation and methylation in ovarian cancer cell lines. ► BRCA1/2 mutations are much less common in cell lines than in the clinic. ► There is a selective pressure against BRCA1/2 mutations in cell culture. ► BRCA1/2 methylated and mutated cell lines are more sensitive to parp inhibitors.
An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence ...or absence of somatic mutations within the patient's tumor that can confer clinical efficacy or drug resistance.
The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development.
Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors.
Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.
We designed and synthesised a series of novel chalcones, incorporating the heterocyclic framework of 2,2-dimethyl-2,3-dihydro-4(1H)-quinolinone, which was prepared via Sonogashira coupling of a ...substituted orthoaniline under aqueous conditions using Pd catalysis followed by acid-mediated cyclisation. The compounds were screened against the NCI-N87 and DLD-1 cancer cell lines, with most compounds showing low micromolar cytotoxic activity.
Current molecular tumor diagnostics encompass panel sequencing to detect mutations, copy number alterations, and rearrangements. However, tumor suppressor genes can also be inactivated by methylation ...within their promoter region. These epigenetic alterations are so far rarely assessed in the clinical setting. Therefore, we established the AllCap protocol facilitating the combined detection of mutations and DNA methylation at the coding and promoter regions of 342 DNA repair genes in one experiment. We demonstrate the use of the protocol by applying it to ovarian cancer cell lines with different responsiveness to poly(ADP-ribose) polymerase inhibition. BRCA1, ATM, ATR, and EP300 mutations and methylation of the BRCA1 promoter were detected as potential predictors for therapy response. The required amount of input DNA was optimized, and the application to formalin-fixed, paraffin-embedded tissue samples was verified to improve the clinical applicability. Thus, by adding DNA methylation values to panel resequencings, the AllCap assay will add another important level of information to clinical tests and will improve stratification of patients for systemic therapies.
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