KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3 using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor

A combined computational and functional approach identifies IGF2BP2 as a driver of chemoresistance in a wide array of pre-clinical models of colorectal cancer

Aim Chemoresistance is a major cause of treatment failure in colorectal cancer (CRC) therapy. In this study, the impact of the IGF2BP family of RNA-binding proteins on CRC chemoresistance was investigated using in silico, in vitro, and in vivo approaches. Methods Gene expression data from a well-characterized cohort and publicly available cross-linking immunoprecipi‑ tation sequencing (CLIP-Seq) data were collected. Resistance to chemotherapeutics was assessed in patient-derived xenografts (PDXs) and patient-derived organoids (PDOs). Functional studies were performed in 2D and 3D cell culture models, including proliferation, spheroid growth, and mitochondrial respiration analyses. Results We identifed IGF2BP2 as the most abundant IGF2BP in primary and metastastatic CRC, correlating with tumor stage in patient samples and tumor growth in PDXs. IGF2BP2 expression in primary tumor tissue was signif‑ cantly associated with resistance to selumetinib, geftinib, and regorafenib in PDOs and to 5-fuorouracil and oxalipl‑ atin in PDX in vivo. IGF2BP2 knockout (KO) HCT116 cells were more susceptible to regorafenib in 2D and to oxaliplatin, selumitinib, and nintedanib in 3D cell culture. Further, a bioinformatic analysis using CLIP data suggested stabiliza‑ tion of target transcripts in primary and metastatic tumors. Measurement of oxygen consumption rate (OCR) and extracellular acidifcation rate (ECAR) revealed a decreased basal OCR and an increase in glycolytic ATP production rate in IGF2BP2 KO. In addition, real-time reverse transcriptase polymerase chain reaction (qPCR) analysis confrmed decreased expression of genes of the respiratory chain complex I, complex IV, and the outer mitochondrial membrane in IGF2BP2 KO cells. Conclusions IGF2BP2 correlates with CRC tumor growth in vivo and promotes chemoresistance by altering mito‑ chondrial respiratory chain metabolism. As a druggable target, IGF2BP2 could be used in future CRC therapy to overcome CRC chemoresistance

Optimización de una estrategia de sondas candado basadas en mRNA

In situ padlock probes experiments are a common tool in single cell genomics. When applied to detect mRNA sequences, they allow quantification and localization of targeted transcript in each individual cell. These assays are mainly performed on cells attached to a glass slide or fixed tissue samples. However, if the padlock probes could be applied on cellular suspensions, the technique could be combined with a wide range of follow-up techniques, such as micromanipulation. In order to test this, in situ padlock probing assays were performed on cellular suspensions and on cell seeded slides. The probes targeted mRNAs for β-actin and androgen receptor full length and were employed in three different cell lines: LNCaP, VCaP and HT-29. The results were observed by fluorescence microscopy and quantified by CellProfiler. In order to test the feasibility of a low coverage genetic sequencing, single cells were selected from the probed cellular suspension via micromanipulation, its DNA was amplified using whole genome amplification and the quality of the amplification product was verified by multiplex PCR. In situ padlock probes assays could be applied to cellular suspensions and expression of the transcripts could be quantified and located in a similar manner to the methods for cells attached onto slides. The number of signals between both supports showed comparable results. Hence any further modifications of the protocol should be focused on improving its efficiency. DNA from probed cells might be of good quality to undergo sequencing. Los ensayos in situ de sondas candado son una herramienta común en genómica de células individuales. Cuando se utilizan para detectar secuencias de mRNA, permiten cuantificar y localizar la expresión del transcrito diana en cada célula individual. Estos ensayos se realizan principalmente en células adheridas a láminas de cristal y en muestras de tejido fijadas, sin embargo, si pudiesen ser aplicadas en células en suspensión, se podrían combinar con un amplio rango de técnicas, como por ejemplo la micromanipulación. Con objetivo de probar esto, se realizaron ensayos in situ de sondas candado en suspensiones celulares y en placas sembradas con células. Las sondas detectaban los mRNA de actina β y del receptor de andrógenos de longitud completa y fueron empleadas en tres líneas celulares distintas: LNCaP, VCaP y HT-29. Los resultados fueron observados en microscopio de fluorescencia y cuantificados usando CellProfiler. Para comprobar la posibilidad de usar una secuenciación de baja cobertura en muestras que hayan sufrido el sondeo, se seleccionaron células individuales mediante micromanipulación, su DNA fue amplificado usando secuenciación de genoma completo y la calidad del producto amplificado fue comprobada por PCR multiplex. El ensayo de sondeo in situ por sondas candado pudo ser aplicado a suspensiones celulares y sus resultados pudieron ser cuantificados y localizados usando métodos similares a aquellos utilizados en células adheridas. El número de señales detectadas mostró resultados comparables entre ambos soportes por lo que cualquier modificación posterior debería centrarse en mejorar su eficiencia. El material genético obtenido de células sondeadas podría tener la calidad suficiente para justificar la secuenciación

Detection of fetal sex, aneuploidy and a microdeletion from single placental syncytial nuclear aggregates

Objectives: A key problem in prenatal screening using extra-embryonic cells is the feasibility of extracting usable DNA from a small number of cells. Syncytial nuclear aggregates (SNAs) are multinucleated structures shed from the placenta. This study assesses the potential of SNAs as a source of fetal DNA for the detection of genetic abnormalities. Methods: SNAs were collected in vitro. Whole-genome amplification was used to amplify DNA from single SNAs, and DNA quality and quantity was assessed by spectrophotometry and PCR. Confocal microscopy was used to count nuclei within SNAs, determine metabolic activity and investigate DNA damage. Fetal sex and chromosomal/genetic abnormalities were investigated with array-comparative genomic hybridization (aCGH). Results: DNA was amplified from 81% of the individual SNAs. A mean of 61 ± 43 nuclei were found per SNA. DNA strand breaks were found in 76% of the SNAs. Seventy-five percent of SNAs yielded whole-genome-amplified DNA of sufficient quality for aCGH after storage and shipping. Individual SNAs from the same pregnancy reliably gave the same chromosomal profile, and fetal sex and trisomies could be detected. A microdeletion was detected in one pregnancy. Conclusion: SNAs could provide a source of extra-embryonic DNA for the prenatal screening/diagnosis of fetal sex and chromosomal and sub-chromosomal genetic abnormalities.</p