MicroRNAs and their roles in breast cancer bone metastasis

Bone metastasis occurs in advanced stages of breast cancer, worsening the quality of life and increasing the mortality of patients. Current treatments for bone metastasis are only palliative, and efficient therapeutic targets need to be still identified. MicroRNAs (miRNAs) are a large class of small non-coding RNAs that regulate gene expression within cells. Interestingly, the expression of certain miRNAs has been associated with several stages of bone metastasis progression, highlighting the importance of these small RNAs during the course of the metastatic disease. In this review, we aim to summarise the most recent findings on miRNAs and their mRNA targets in driving breast cancer bone metastasis. Furthermore, we discuss the possibility to use miRNAs as direct therapeutic targets or as advanced therapies for breast cancer bone metastasis, as well as their potential as predictive biomarkers of bone metastasis for an early diagnosis and a better tailoring of therapies for cancer patients

Debunking the myth of the endogenous antiangiogenic Vegfaxxxb transcripts

In this opinion article we critically assess evidence for the existence of a family of antiangiogenic vascular endothelial growth factor (Vegfaxxxb) transcripts, arising from the use of a phylogenetically conserved alternative distal splice site within exon 8 of the VEGFA gene. We explain that prior evidence for Vegfaxxxb transcripts in tissues rests heavily upon flawed RT-PCR methodologies, with the extensive use of 5′-tailing in primer design being the main issue. Furthermore, our analysis of large RNA-seq data sets (human and ovine) fails to identify a single Vegfaxxxb transcript. Therefore, we challenge the very existence of Vegfaxxxb transcripts, which further questions the physiological relevance of studies based on the use of ‘anti-VEGFAxxxb’ antibodies. Our analysis has implications for the proposed therapeutic use of isoform-specific anti-VEGFA strategies for treating cancer and retinopathies

Increased non-homologous end joining makes DNA-PK a promising target for therapeutic intervention in uveal melanoma

Uveal melanoma (UM) is the most common primary intraocular tumour in adults, with a mean survival of six months following metastasis. The survival rates have not improved in over 30 years. This study has shown that sister chromatid exchange (SCE) is low in UM which is likely due to a reduced expression of FANCD2. As FANCD2 can function to suppress non-homologous end joining (NHEJ), this study therefore investigated NHEJ in UM. The activation of the catalytic subunit of the NHEJ pathway protein DNA-dependent protein kinase (DNA-PK) was measured by analysing the foci formation and the ligation efficiency by NHEJ determined using a plasmid-based end-joining assay. Using small-interfering RNA (siRNA) knock-down, and chemical inhibitors of DNA-PK, the survival of primary UM cultures and two cell lines were determined. To assess the homologous recombination capacity in response to the inhibition of DNA-PK, a SCE analysis was performed. In addition, to support the findings, the messenger RNA (mRNA) expression of genes associated with NHEJ was analysed using the Cancer Genome Atlas (TCGA)-UM RNAseq data (n = 79). The NHEJ activity and DNA-PKcs activation was upregulated in UM and the inhibition of DNA-PK selectively induced apoptosis and sensitized to ionising radiation and inter-strand cross-linking agents. The inhibition of the NHEJ protein DNA-PK is lethal to UM, indicating a potentially effective therapeutic option, either alone or as a sensitizer for other treatments

Biallelic inheritance of hypomorphic PKD1 variants is highly prevalent in very early onset polycystic kidney disease

Purpose To investigate the prevalence of biallelic PKD1 and PKD2 variants underlying very early onset (VEO) polycystic kidney disease (PKD) in a large international pediatric cohort referred for clinical indications over a 10-year period (2010–2020). Methods All samples were tested by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) of PKD1 and PKD2 genes and/or a next-generation sequencing panel of 15 additional cystic genes including PKHD1 and HNF1B. Two patients underwent exome or genome sequencing. Results Likely causative PKD1 or PKD2 variants were detected in 30 infants with PKD-VEO, 16 of whom presented in utero. Twenty-one of 30 (70%) had two variants with biallelic in trans inheritance confirmed in 16/21, 1 infant had biallelic PKD2 variants, and 2 infants had digenic PKD1/PKD2 variants. There was no known family history of ADPKD in 13 families (43%) and a de novo pathogenic variant was confirmed in 6 families (23%). Conclusion We report a high prevalence of hypomorphic PKD1 variants and likely biallelic disease in infants presenting with PKD-VEO with major implications for reproductive counseling. The diagnostic interpretation and reporting of these variants however remains challenging using current American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) and Association of Clinical Genetic Science (ACGS) variant classification guidelines in PKD-VEO and other diseases affected by similar variants with incomplete penetrance

The positive effect of selective prostaglandin E2 receptor EP2 and EP4 blockade on cystogenesis in vitro is counteracted by increased kidney inflammation in vivo

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major cause of end-stage kidney disease in man. The central role of cyclic adenosine monophosphate (cAMP) in ADPKD pathogenesis has been confirmed by numerous studies including positive clinical trial data. Here, we investigated the potential role of another major regulator of renal cAMP, prostaglandin E2 (PGE2), in modifying disease progression in ADPKD models using selective receptor modulators to all four PGE2 receptor subtypes (EP1-4). In 3D-culture model systems utilizing dog (MDCK) and patient-derived (UCL93, OX161-C1) kidney cell lines, PGE2 strikingly promoted cystogenesis and inhibited tubulogenesis by stimulating proliferation while reducing apoptosis. The effect of PGE2 on tubulogenesis and cystogenesis in 3D-culture was mimicked or abolished by selective EP2 and EP4 agonists or antagonists but not those specific to EP1 or EP3. In a Pkd1 mouse model (Pkd1nl/nl), kidney PGE2 and COX-2 expression were increased by two-fold at the peak of disease (week four). However, Pkd1nl/nl mice treated with selective EP2 (PF-04418948) or EP4 (ONO-AE3-208) antagonists from birth for three weeks had more severe cystic disease and fibrosis associated with increased cell proliferation and macrophage infiltration. A similar effect was observed for the EP4 antagonist ONO-AE3-208 in a second Pkd1 model (Pax8rtTA-TetO-Cre-Pkd1f/f). Thus, despite the positive effects of slowing cyst growth in vitro, the more complex effects of inhibiting EP2 or EP4 in vivo resulted in a worse outcome, possibly related to unexpected pro-inflammatory effects

MiR-662 is associated with metastatic relapse in early-stage breast cancer and promotes metastasis by stimulating cancer cell stemness

Background Breast cancer (BC) metastasis, which often occurs in bone, contributes substantially to mortality. MicroRNAs play a fundamental role in BC metastasis, although microRNA-regulated mechanisms driving metastasis progression remain poorly understood. Methods MiRome analysis in serum from BC patients was performed by TaqMan™ low-density array. MiR-662 was overexpressed following MIMIC-transfection or lentivirus transduction. Animal models were used to investigate the role of miR-662 in BC (bone) metastasis. The effect of miR-662-overexpressing BC cell conditioned medium on osteoclastogenesis was investigated. ALDEFLUOR assays were performed to study BC stemness. RNA-sequencing transcriptomic analysis of miR-662-overexpressing BC cells was performed to evaluate gene expression changes. Results High levels of hsa-miR-662 (miR-662) in serum from BC patients, at baseline (time of surgery), were associated with future recurrence in bone. At an early-stage of the metastatic disease, miR-662 could mask the presence of BC metastases in bone by inhibiting the differentiation of bone-resorbing osteoclasts. Nonetheless, metastatic miR-662-overexpressing BC cells then progressed as overt osteolytic metastases thanks to increased stem cell-like traits. Conclusions MiR-662 is involved in BC metastasis progression, suggesting it may be used as a prognostic marker to identify BC patients at high risk of metastasis

Global microRNA profiling in human urinary exosomes reveals novel disease biomarkers and cellular pathways for Autosomal Dominant Polycystic Kidney Disease

MicroRNAs (miRNAs) play an important role in regulating gene expression in health and disease but their role in modifying disease expression in Autosomal Dominant Polycystic Kidney Disease (ADPKD) remains uncertain. Here, we profiled human urinary exosome miRNA by global small RNA-sequencing in an initial discovery cohort of seven patients with ADPKD with early disease (eGFR over 60ml/min/1.73m2), nine with late disease (eGFR under 60ml/min/1.73m2), and compared their differential expression with six age and sex matched healthy controls. Two kidney-enriched candidate miRNA families were identified (miR-192/miR-194-2 and miR-30) and selected for confirmatory testing in a 60 patient validation cohort by quantitative polymerase chain reaction. We confirmed that miR-192-5p, miR-194- 5p, miR-30a-5p, miR-30d-5p and miR-30e-5p were significantly downregulated in patient urine exosomes, in murine Pkd1 cystic kidneys and in human PKD1 cystic kidney tissue. All five miRNAs showed significant correlations with baseline eGFR and ultrasound-determined mean kidney length and improved the diagnostic performance (area under the curve) of mean kidney length for the rate of disease progression. Finally, inverse correlations of these two miRNA families with increased expression in their predicted target genes in patient PKD1 cystic tissue identified dysregulated pathways and transcriptional networks including novel interactions between miR-194-5p and two potentially relevant candidate genes, PIK3R1 and ANO1. Thus, our results identify a subset of urinary exosomal miRNAs that could serve as novel biomarkers of disease progression and suggest new therapeutic targets in ADPKD

The common PKD1 p.(Ile3167Phe) variant is hypomorphic and associated with very early onset, biallelic polycystic kidney disease

Biallelic PKD1 variants, including hypomorphic variants, can cause very early onset polycystic kidney disease (VEO-PKD). A family with unexplained recurrent VEO-PKD and neonatal demise in one dizygotic twin was referred for clinical testing. Further individuals with the putative hypomorphic PKD1 variant, p.(Ile3167Phe), were identified from the UK 100,000 genomes project (100 K), UK Biobank (UKBB), and a review of the literature. We identified a likely pathogenic PKD1 missense paternal variant and the putative hypomorphic PKD1 variant from the unaffected mother in the deceased twin but only the paternal PKD1 variant in the surviving dizygotic twin. Analysis of 100 K cases identified a second family with two siblings with similar biallelic inheritance who presented at birth with VEO-PKD and reached kidney failure in their teens unlike other affected relatives. Finally, a survey of 618 UKBB cases confirmed that adult patients monoallelic for PKD1 p.(Ile3167Phe) had normal kidney function. Our data reveals that p.(Ile3167Phe) is the second most common PKD1 hypomorphic variant identified and is neutral in heterozygosity but is associated with VEO-PKD when inherited in trans with a pathogenic PKD1 variant. Care should be taken to ensure that it is not automatically filtered from sequence data for VEO cases

Combining genotype with height-adjusted kidney length predicts rapid progression of ADPKD

Introduction Our main objective was to identify baseline prognostic factors predictive of rapid disease progression in a large unselected clinical autosomal dominant polycystic kidney disease (ADPKD) cohort. Methods A cross-sectional analysis was performed in 618 consecutive ADPKD patients assessed and followed-up for over a decade. A total of 123 patients (19.9%) had reached kidney failure by the study date. Data were available for the following: baseline eGFR (n = 501), genotype (n = 549), baseline ultrasound mean kidney length (MKL, n = 424) and height-adjusted baseline MKL (HtMKL, n = 377). Rapid disease progression was defined as an annualized eGFR decline (∆eGFR) of >2.5 mL/min/year by linear regression over 5 years (n = 158). Patients were further divided into slow, rapid and very rapid ∆eGFR classes for analysis. Genotyped patients were classified into several categories: PKD1 (T, truncating; or NT, non-truncating), PKD2, other genes (non-PKD1 or -PKD2), no mutation detected or variants of uncertain significance. Results A PKD1-T genotype had the strongest influence on the probability of reduced baseline kidney function by age. A multivariate logistic regression model identified PKD1-T genotype and HtMKL (>9.5 cm/m) as independent predictors for rapid disease progression. The combination of both factors increased the positive predictive value for rapid disease progression over age 40 years and of reaching kidney failure by age 60 years to 100%. Exploratory analysis in a subgroup with available total kidney volumes showed higher positive predictive value (100% vs 80%) and negative predictive value (42% vs 33%) in predicting rapid disease progression compared with the Mayo Imaging Classification (1C–E). Conclusion Real-world longitudinal data confirm the importance of genotype and kidney length as independent variables determining ∆eGFR. Individuals with the highest risk of rapid disease progression can be positively selected for treatment based on this combination

A founder UMOD variant is a common cause of hereditary nephropathy in the British population

Background Monogenic disorders are estimated to account for 10%–12% of patients with kidney failure. We report the unexpected finding of an unusual uromodulin (UMOD) variant in multiple pedigrees within the British population and demonstrate a shared haplotype indicative of an ancestral variant. Methods Probands from 12 apparently unrelated pedigrees with a family history of kidney failure within a geographically contiguous UK region were shown to be heterozygous for a pathogenic variant of UMOD c.278_289delTCTGCCCCGAAG insCCGCCTCCT. Results A total of 88 clinically affected individuals were identified, all born in the UK and of white British ethnicity. 20 other individuals with the variant were identified in the UK 100,000 Genomes (100K) Project and 9 from UK Biobank (UKBB). A common extended haplotype was present in 5 of the UKBB individuals who underwent genome sequencing which was only present in <1 in 5000 of UKBB controls. Significantly, rare variants (<1 in 250 general population) identified within 1 Mb of the UMOD variant by genome sequencing were detected in all of the 100K individuals, indicative of an extended shared haplotype. Conclusion Our data confirm a likely founder UMOD variant with a wide geographical distribution within the UK. It should be suspected in cases of unexplained familial nephropathy presenting in patients of white British ancestry