Response to : The GPRC5A frameshift variant c.183del is not associated with increased breast cancer risk in BRCA1 mutation carriers

Non peer reviewe

BRCA1-deficient breast cancer cell lines are resistant to MEK inhibitors and show distinct sensitivities to 6-thioguanine

Germ-line or somatic inactivation of BRCA1 is a defining feature for a portion of human breast cancers. Here we evaluated the anti-proliferative activity of 198 FDA-approved and experimental drugs against four BRCA1-mutant (HCC1937, MDA-MB-436, SUM1315MO2, and SUM149PT) and four BRCA1-wild-type (MDA-MB-231, SUM229PE, MCF10A, and MCF7) breast cancer cell lines. We found that all BRCA1-mutant cell lines were insensitive to inhibitors of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) Selumetinib and Pimasertib in contrast to BRCA1-wildtype control cell lines. However, unexpectedly, only two BRCA1-mutant cell lines, HCC1937 and MDA-MB-436, were hypersensitive to a nucleotide analogue 6-thioguanine (6-TG). SUM149PT cells readily formed radiation-induced RAD51-positive nuclear foci indicating a functional homologous recombination, which may explain their resistance to 6-TG. However, the reason underlying 6-TG resistance of SUM1315MO2 cells remains unclear. Our data reveal a remarkable heterogeneity among BRCA1-mutant cell lines and provide a reference for future studies.Peer reviewe

Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion

G-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin β1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.</p

Adaptive RSK-EphA2-GPRC5A signaling switch triggers chemotherapy resistance in ovarian cancer

Metastatic cancers commonly activate adaptive chemotherapy resistance, attributed to both microenvironment-dependent phenotypic plasticity and genetic characteristics of cancer cells. However, the contribution of chemotherapy itself to the non-genetic resistance mechanisms was long neglected. Using high-grade serous ovarian cancer (HGSC) patient material and cell lines, we describe here an unexpectedly robust cisplatin and carboplatin chemotherapy-induced ERK1/2-RSK1/2-EphA2-GPRC5A signaling switch associated with cancer cell intrinsic and acquired chemoresistance. Mechanistically, pharmacological inhibition or knockdown of RSK1/2 prevented oncogenic EphA2-S897 phosphorylation and EphA2-GPRC5A co-regulation, thereby facilitating a signaling shift to the canonical tumor-suppressive tyrosine phosphorylation and consequent downregulation of EphA2. In combination with platinum, RSK inhibitors effectively sensitized even the most platinum-resistant EphA2(high), GPRC5A(high) cells to the therapy-induced apoptosis. In HGSC patient tumors, this orphan receptor GPRC5A was expressed exclusively in cancer cells and associated with chemotherapy resistance and poor survival. Our results reveal a kinase signaling pathway uniquely activated by platinum to elicit adaptive resistance. They further identify GPRC5A as a marker for abysmal HGSC outcome and putative vulnerability of the chemo-resistant cells to RSK1/2-EphA2-pS897 pathway inhibition.Peer reviewe

Chemical, Physical and Biological Triggers of Evolutionary Conserved Bcl-xL-Mediated Apoptosis

Background: The evidence that pan-Bcl-2 or Bcl-xL-specific inhibitors prematurely kill virus-infected or RNA/DNA-transfected cells provides rationale for investigating these apoptotic inducers further. We hypothesized that not only invasive RNA or DNA (biological factors) but also DNA/RNA-damaging chemical or physical factors could trigger apoptosis that have been sensitized with pan-Bcl-2 or Bcl-xL-specific agents; Methods: We tested chemical and physical factors plus Bcl-xL-specific inhibitor A-1155463 in cells of various origins and the small roundworms (C. elegans); Results: We show that combination of a A-1155463 along with a DNA-damaging agent, 4-nitroquinoline-1-oxide (4NQO), prematurely kills cells of various origins as well as C. elegans. The synergistic effect is p53-dependent and associated with the release of Bad and Bax from Bcl-xL, which trigger mitochondrial outer membrane permeabilization. Furthermore, we found that combining Bcl-xL-specific inhibitors with various chemical compounds or physical insults also induced cell death; Conclusions: Thus, we were able to identify several biological, chemical and physical triggers of the evolutionarily conserved Bcl-xL-mediated apoptotic pathway, shedding light on strategies and targets for novel drug development

Network-guided identification of cancer-selective combinatorial therapies in ovarian cancer

Each patient's cancer consists of multiple cell subpopulations that are inherently heterogeneous and may develop differing phenotypes such as drug sensitivity or resistance. A personalized treatment regimen should therefore target multiple oncoproteins in the cancer cell populations that are driving the treatment resistance or disease progression in a given patient to provide maximal therapeutic effect, while avoiding severe co-inhibition of non-malignant cells that would lead to toxic side effects. To address the intra- and inter-tumoral heterogeneity when designing combinatorial treatment regimens for cancer patients, we have implemented a machine learning-based platform to guide identification of safe and effective combinatorial treatments that selectively inhibit cancer-related dysfunctions or resistance mechanisms in individual patients. In this case study, we show how the platform enables prediction of cancer-selective drug combinations for patients with high-grade serous ovarian cancer using single-cell imaging cytometry drug response assay, combined with genome-wide transcriptomic and genetic profiles. The platform makes use of drug-target interaction networks to prioritize those combinations that warrant further preclinical testing in scarce patient-derived primary cells. During the case study in ovarian cancer patients, we investigated (i) the relative performance of various ensemble learning algorithms for drug response prediction, (ii) the use of matched single-cell RNA-sequencing data to deconvolute cell population-specific transcriptome profiles from bulk RNA-seq data, (iii) and whether multi-patient or patient-specific predictive models lead to better predictive accuracy. The general platform and the comparison results are expected to become useful for future studies that use similar predictive approaches also in other cancer types.</p

Loss of DIAPH1 causes SCBMS, combined immunodeficiency, and mitochondrial dysfunction

Background: Homozygous loss of DIAPH1 results in seizures, cortical blindness, and microcephaly syndrome (SCBMS). We studied 5 Finnish and 2 Omani patients with loss of DIAPH1 presenting with SCBMS, mitochondrial dysfunction, and immunodeficiency. Objective: We sought to further characterize phenotypes and disease mechanisms associated with loss of DIAPH1. Methods: Exome sequencing, genotyping and haplotype analysis, B- and T-cell phenotyping, in vitro lymphocyte stimulation assays, analyses of mitochondrial function, immunofluorescence staining for cytoskeletal proteins and mitochondria, and CRISPR-Cas9 DIAPH1 knockout in heathy donor PBMCs were used. Results: Genetic analyses found all Finnish patients homozygous for a rare DIAPH1 splice-variant (NM_005219:c.68411G>A) enriched in the Finnish population, and Omani patients homozygous for a previously described pathogenic DIAPH1 frameshift-variant (NM_005219:c.2769delT;p.F923fs). In addition to microcephaly, epilepsy, and cortical blindness characteristic to SCBMS, the patients presented with infection susceptibility due to defective lymphocyte maturation and 3 patients developed B-cell lymphoma. Patients' immunophenotype was characterized by poor lymphocyte activation and proliferation, defective B-cell maturation, and lack of naive T cells. CRISPR-Cas9 knockout of DIAPH1 in PBMCs from healthy donors replicated the T-cell activation defect. Patient-derived peripheral blood T cells exhibited impaired adhesion and inefficient microtubule-organizing center repositioning to the immunologic synapse. The clinical symptoms and laboratory tests also suggested mitochondrial dysfunction. Experiments with immortalized, patient-derived fibroblasts indicated that DIAPH1 affects the amount of complex IV of the mitochondrial respiratory chain. Conclusions: Our data demonstrate that individuals with SCBMS can have combined immune deficiency and implicate defective cytoskeletal organization and mitochondrial dysfunction in SCBMS pathogenesis.Peer reviewe

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.Peer reviewe

Antiviral properties of chemical inhibitors of cellular anti-apoptotic Bcl-2 proteins

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.</p

Uudet geneettiset riskitekijät rintasyövän kehityksessä

Breast cancer is the most common malignancy in women. Familial history and inherited genetic mutations are the most important risk factors for the development of this type of neoplasia. However, according to current estimations, known highly penetrant germline mutations (e.g. in BRCA1 and BRCA2 genes) explain the origin of less than 30% of familial breast cancer cases 2 5. It urges for identification of the novel genetic determinants of breast carcinogenesis. In the presented work, we performed a whole exome sequencing-based search for new breast cancer-predisposing genetic mutations. We identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor-encoding gene GPRC5A as a novel genetic determinant of breast cancer, which is highly prevalent in BRCA1-associated familial tumors. Following this finding, we revealed that GPRC5A modulates BRCA1 expression and function in homologous recombination-mediated DNA repair, suggesting that GPRC5A may act as a genetic modifier of BRCA1-mediated breast cancer progression. GPRC5A has been implicated in the epithelial carcinogenesis, but so far its function remains controversial. Exploring the effect of GPRC5A deficiency or excessive expression on different aspects of cancer progression, we discovered that GPRC5A plays a role in cancer cell-matrix interactions, and found that it functions as a positive modulator of integrin β1-mediated adhesion. The data suggest that GPRC5A may play a role in progression of particular types of epithelial tumors. We found that GPRC5A associated with two more matrix interaction regulators, Ephrin receptor A2 (EphA2) and hyaluronic acid receptor RHAMM; the effect of GPRC5A on RHAMM expression was BRCA1-dependent. Finally, we revealed that GPRC5A expression in mammary glands is estrogen-dependent and correlates with proliferative capacity of estrogen receptor-positive (ER+) mammary tumors, proposing GPRC5A as a biomarker of ER+ tumors progression. We propose that GPRC5A is a determinant of breast carcinogenesis, whose function depends on the context of the genetic background (presence of BRCA1 mutation) and the hormonal status of the tumor.Naisten yleisin syöpä on rintasyöpä 1. Tärkeimpiä rintasyövän riskitekijöitä ovat suvun syöpähistoria ja perinnölliset geneettiset mutaatiot. Nykyisten arvioiden mukaan yleisimmät perinnölliset mutaatiot (esim. BRCA1- ja BRCA2-geeneissä) selittävät kuitenkin vain n. 30 % perinnöllisistä rintasyöpätapauksista 2-5. Uusien rintasyövälle altistavien geneettisten tekijöiden löytäminen on näin ollen tärkeää. Tässä työssä käytimme eksomisekvensointia löytääksemme uusia rintasyövälle altistavia geneettisiä mutaatioita. Löysimme inaktivoivan mutaation c.183delG [p. Arg61fs] geenistä, joka koodaa G-proteiinikytköksistä reseptoriproteiinia GPRC5A. Tämä uusi rintasyövän geneettinen determinantti on erityisen yleinen BRCA1-mutatoituneissa perinnöllisissä syövissä. Me saimme selville, että GPRC5A säätelee BRCA1:n ilmentymistä ja toimintaa homologiseen rekombinaatioon perustuvassa DNA-korjauksessa. GPRC5A:n toimintaa on aiemmin tutkittu epiteelisolujen karsinogeneesissä, mutta sen rooli on vielä kiistanalainen. Yli- ja ali-ekspressoimalla GPRC5A:a saimme selville että GPRC5A:lla on rooli syöpäsolujen ja solujenvälisen matriksin vuorovaikutuksissa, joissa se lisää integriini β1-välitteistä soluadheesiota. Tulostemme mukaan GPRC5A saattaa myös olla erityisen tärkeä tietynlaisten epiteelisyöpien kehityksessä. GPRC5A voidaan yhdistää integriini β1:n lisäksi kahteen muuhun matriksi-interaktion säätelijään, efriini-reseptori 2A:han ja hyaluronihapporeseptori RHAMM:iin. Totesimme GPRC5A:n säätelevän RHAMM:in ilmentymistä BRCA1-riippuvaisesti. Tuloksemme paljastivat myös, että GPRC5A-ekspressio rintarauhasissa on riippuvaista estrogeenista ja korreloi estrogeeni-reseptori-positiivisten (ER+) rintakasvaimien solunjakaantumispotentiaalin kanssa, minkä vuoksi GPCR5A on mahdollinen biomarkkeri ER+ kasvaimien kehityksessä. Me ehdotamme, että GPRC5A, jonka rooli riippuu geneettisestä ympäristöstä (mutaatio BRCA1:ssä) ja kasvaimen hormonistatuksesta, on tärkeä tekijä rintakudoksen karsinogeneesissä.Julkaistu painettuna: Helsinki: Hansaprint, 2016. Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis, 2342-316