The drug efficacy testing in 3D cultures platform identifies effective drugs for ovarian cancer patients.

Most patients with advanced ovarian cancer (OC) relapse and progress despite systemic therapy, pointing to the need for improved and tailored therapy options. Functional precision medicine can help to identify effective therapies for individual patients in a clinically relevant timeframe. Here, we present a scalable functional precision medicine platform: DET3Ct (Drug Efficacy Testing in 3D Cultures), where the response of patient cells to drugs and drug combinations are quantified with live-cell imaging. We demonstrate the delivery of individual drug sensitivity profiles in 20 samples from 16 patients with ovarian cancer in both 2D and 3D culture formats, achieving over 90% success rate in providing results six days after operation. In this cohort all patients received carboplatin. The carboplatin sensitivity scores were significantly different for patients with a progression free interval (PFI) less than or equal to 12 months and those with more than 12 months (p < 0.05). We find that the 3D culture format better retains proliferation and characteristics of the in vivo setting. Using the DET3Ct platform we evaluate 27 tailored combinations with results available 10 days after operation. Notably, carboplatin and A-1331852 (Bcl-xL inhibitor) showed an additive effect in four of eight OC samples tested, while afatinib and A-1331852 led to synergy in five of seven OC models. In conclusion, our 3D DET3Ct platform can rapidly define potential, clinically relevant data on efficacy of existing drugs in OC for precision medicine purposes, as well as provide insights on emerging drugs and drug combinations that warrant testing in clinical trials.

Multiomics characterization implicates PTK7 in ovarian cancer EMT and cell plasticity and offers strategies for therapeutic intervention.

Most patients with ovarian cancer (OC) are diagnosed at a late stage when there are very few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or an oncogenic addiction that can be targeted pharmacologically, unlike other types of cancer. Here, we identified protein tyrosine kinase 7 (PTK7) as a potential new therapeutic target in OC following a multiomics approach using genetic and pharmacological interventions. We performed proteomics analyses upon PTK7 knockdown in OC cells and identified novel downstream effectors such as synuclein-γ (SNCG), SALL2, and PP1γ, and these findings were corroborated in ex vivo primary samples using PTK7 monoclonal antibody cofetuzumab. Our phosphoproteomics analyses demonstrated that PTK7 modulates cell adhesion and Rho-GTPase signaling to sustain epithelial-mesenchymal transition (EMT) and cell plasticity, which was confirmed by high-content image analysis of 3D models. Furthermore, using high-throughput drug sensitivity testing (525 drugs) we show that targeting PTK7 exhibited synergistic activity with chemotherapeutic agent paclitaxel, CHK1/2 inhibitor prexasertib, and PLK1 inhibitor GSK461364, among others, in OC cells and ex vivo primary samples. Taken together, our study provides unique insight into the function of PTK7, which helps to define its role in mediating aberrant Wnt signaling in ovarian cancer.

PFKFB3 Inhibition Sensitizes DNA Crosslinking Chemotherapies by Suppressing Fanconi Anemia Repair.

Replicative repair of interstrand crosslinks (ICL) generated by platinum chemotherapeutics is orchestrated by the Fanconi anemia (FA) repair pathway to ensure resolution of stalled replication forks and the maintenance of genomic integrity. Here, we identify novel regulation of FA repair by the cancer-associated glycolytic enzyme PFKFB3 that has functional consequences for replication-associated ICL repair and cancer cell survival. Inhibition of PFKFB3 displays a cancer-specific synergy with platinum compounds in blocking cell viability and restores sensitivity in treatment-resistant models. Notably, the synergies are associated with DNA-damage-induced chromatin association of PFKFB3 upon cancer transformation, which further increases upon platinum resistance. FA pathway activation triggers the PFKFB3 assembly into nuclear foci in an ATR- and FANCM-dependent manner. Blocking PFKFB3 activity disrupts the assembly of key FA repair factors and consequently prevents fork restart. This results in an incapacity to replicate cells to progress through S-phase, an accumulation of DNA damage in replicating cells, and fork collapse. We further validate PFKFB3-dependent regulation of FA repair in ex vivo cultures from cancer patients. Collectively, targeting PFKFB3 opens up therapeutic possibilities to improve the efficacy of ICL-inducing cancer treatments.

siRNA Knockdown of REDD1 Facilitates Aspirin-Mediated Dephosphorylation of mTORC1 Target 4E-BP1 in MDA-MB-468 Human Breast Cancer Cell Line.

BACKGROUND: Mutations within genes encoding components of the PI3K/AKT/mTOR (phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin) signaling axis frequently activate the pathway in breast cancer, making it an attractive therapeutic target. Inhibition of mTORC1 (mechanistic target of rapamycin complex 1) activity upon aspirin treatment has been reported in breast cancer cells harboring PI3KCA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation and is considered to account for anticancer action. METHODS: MDA-MB-468 (harbors mutated PTEN (phosphatase and TENsin homolog)), MCF-7 (PI3KCA-mutated), MDA-MB-231 (no PI3K pathway mutations) cancer cell lines and MCF10A non-cancerous breast epithelial cells were employed for the assessment of modulation of mTORC1 signaling by aspirin. Targeted amplicon-based next-generation sequencing using the Ion Torrent technology was carried out to determine gene expression changes following drug treatment. Western blot was performed to analyze the expression and phosphorylation of proteins. Knockdown by siRNA approach was applied to assess the role of REDD1/DDIT4 (DNA damage-inducible transcript 4) in mTORC1 inhibition by aspirin. RESULTS: We show a decline in phosphorylation of mTORC1 downstream substrate 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) in response to treatment with aspirin and its metabolite salicylic acid in MDA-MB-468, MCF-7, MDA-MB-231, and MCF10A cell lines. We further demonstrate a novel molecular response to aspirin in breast cancer cells. Specifically, we found that aspirin and salicylic acid increase the expression of REDD1 protein, that is known for its suppressive function towards mTORC1. Unexpectedly, we observed that siRNA knockdown of REDD1 expression facilitated aspirin-mediated suppression of mTORC1 downstream substrate 4E-BP1 phosphorylation in the MDA-MB-468 cell line. REDD1 downregulation slightly encouraged reduction in 4E-BP1 phosphorylation by aspirin in MCF-7 cells but did not elicit a reproducible effect in the MDA-MB-231 cell line. siRNA knockdown of REDD1 did not affect the expression of phosphorylated form of 4E-BP1 following aspirin treatment in MCF10A non-cancerous breast epithelial cells. CONCLUSION: The current findings suggest that REDD1 downregulation might improve the anticancer activity of aspirin in a subset of breast tumors.

The Role of miR-375-3p and miR-200b-3p in Gastrointestinal Stromal Tumors.

Deregulated microRNA (miRNA) expression profiles and their contribution to carcinogenesis have been observed in virtually all types of human cancer. However, their role in the pathogenesis of rare mesenchymal gastrointestinal stromal tumors (GISTs) is not well defined, yet. In this study, we aimed to investigate the role of two miRNAs strongly downregulated in GIST-miR-375-3p and miR-200b-3p-in the pathogenesis of GIST. To achieve this, miRNA mimics were transfected into GIST-T1 cells and changes in the potential target gene mRNA and protein expression, as well as alterations in cell viability, migration, apoptotic cell counts and direct miRNA-target interaction, were evaluated. Results revealed that overexpression of miR-375-3p downregulated the expression of KIT mRNA and protein by direct binding to KIT 3'UTR, reduced GIST cell viability and migration rates. MiR-200b-3p lowered expression of ETV1 protein, directly targeted and lowered expression of EGFR mRNA and protein, and negatively affected cell migration rates. To conclude, the present study identified that miR-375-3p and miR-200b-3p have a tumor-suppressive role in GIST.