Steering research on mRNA splicing in cancer towards clinical translation.

Splicing factors are affected by recurrent somatic mutations and copy number variations in several types of haematologic and solid malignancies, which is often seen as prima facie evidence that splicing aberrations can drive cancer initiation and progression. However, numerous spliceosome components also 'moonlight' in DNA repair and other cellular processes, making their precise role in cancer difficult to pinpoint. Still, few would deny that dysregulated mRNA splicing is a pervasive feature of most cancers. Correctly interpreting these molecular fingerprints can reveal novel tumour vulnerabilities and untapped therapeutic opportunities. Yet multiple technological challenges, lingering misconceptions, and outstanding questions hinder clinical translation. To start with, the general landscape of splicing aberrations in cancer is not well defined, due to limitations of short-read RNA sequencing not adept at resolving complete mRNA isoforms, as well as the shallow read depth inherent in long-read RNA-sequencing, especially at single-cell level. Although individual cancer-associated isoforms are known to contribute to cancer progression, widespread splicing alterations could be an equally important and, perhaps, more readily actionable feature of human cancers. This is to say that in addition to 'repairing' mis-spliced transcripts, possible therapeutic avenues include exacerbating splicing aberration with small-molecule spliceosome inhibitors, targeting recurrent splicing aberrations with synthetic lethal approaches, and training the immune system to recognize splicing-derived neoantigens.

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia.

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B- ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia.

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B-ALL patients, suggesting additional mechanisms of resistance. By mining RNA sequencing datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, antifolates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-mercaptopurine in B-ALL cells both in vitro and in vivo. Furthermore, both NT5C2ex6a and the R238W variant induced collateral sensitivity to the inosine monophosphate dehydrogenase inhibitor mizoribine. These results ascribe to splicing perturbations an important role in chemotherapy resistance in relapsed B-ALL and suggest that inosine monophosphate dehydrogenase inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias. Significance: Alternative splicing is a potent mechanism of acquired drug resistance in relapsed/refractory acute lymphoblastic leukemias that has diagnostic and therapeutic implications for patients who lack mutations in known chemoresistance genes.

Mapping and modeling human colorectal carcinoma interactions with the tumor microenvironment.

The initiation and progression of cancer are intricately linked to the tumor microenvironment (TME). Understanding the function of specific cancer-TME interactions poses a major challenge due in part to the complexity of the in vivo microenvironment. Here we predict cancer-TME interactions from single cell transcriptomic maps of both human colorectal cancers (CRCs) and mouse CRC models, ask how these interactions are altered in human tumor organoid (tumoroid) cultures, and functionally recapitulate human myeloid-carcinoma interactions in vitro. Tumoroid cultures suppress gene expression programs involved in inflammation and immune cell migration, providing a reductive platform for re-establishing carcinoma-immune cell interactions in vitro. Introduction of human monocyte-derived macrophages into tumoroid cultures instructs macrophages to acquire immunosuppressive and pro-tumorigenic gene expression programs similar to those observed in vivo. This includes hallmark induction of SPP1, encoding Osteopontin, an extracellular CD44 ligand with established oncogenic effects. Taken together, these findings offer a framework for understanding CRC-TME interactions and provide a reductionist tool for modeling specific aspects of these interactions.

MYC Hyperactivates Wnt Signaling in APC/CTNNB1-Mutated Colorectal Cancer Cells through miR-92a-Dependent Repression of DKK3.

Activation of Wnt signaling is among the earliest events in colon cancer development. It is achieved either via activating mutations in the CTNNB1 gene encoding ß-catenin, the key transcription factor in the Wnt pathway, or most commonly by inactivating mutations affecting APC, a major ß-catenin binding partner and negative regulator. However, our analysis of recent Pan Cancer Atlas data revealed that CTNNB1 mutations significantly co-occur with those affecting Wnt receptor complex components (e.g., Frizzled and LRP6), underscoring the importance of additional regulatory events even in the presence of common APC/CTNNB1 mutations. In our effort to identify non-mutational hyperactivating events, we determined that KRAS-transformed murine colonocytes overexpressing direct ß-catenin target MYC show significant upregulation of the Wnt signaling pathway and reduced expression of Dickkopf 3 (DKK3), a reported ligand for Wnt co-receptors. We demonstrate that MYC suppresses DKK3 transcription through one of miR-17-92 cluster miRNAs, miR-92a. We further examined the role of DKK3 by overexpression and knockdown and discovered that DKK3 suppresses Wnt signaling in Apc-null murine colonic organoids and human colon cancer cells despite the presence of downstream activating mutations in the Wnt pathway. Conversely, MYC overexpression in the same cell lines resulted in hyperactive Wnt signaling, acquisition of epithelial-to-mesenchymal transition markers, and enhanced migration/invasion in vitro and metastasis in a syngeneic orthotopic mouse colon cancer model. IMPLICATIONS: Our results suggest that the MYC→miR-92a-|DKK3 axis hyperactivates Wnt signaling, forming a feed-forward oncogenic loop.

Does maternal tooth brushing-related sef-efficacy predict child's brushing adherence?

BACKGROUND: Dental plaque is a root cause of dental caries. Effective plaque control in young children can be achieved with twice-daily assisted tooth brushing. Self-efficacy relates to one's confidence in performing a task. Self-efficacy is shown to facilitate the behavior change in treatments for lifestyle diseases. The influence of maternal self-efficacy in children's oral health behaviors is less studied. AIM: The aim of this study is to evaluate an association between maternal tooth brushing-related self-efficacy (MTBSE) and child's brushing adherence. SETTINGS AND DESIGN: This cross-sectional study was conducted in schools and included 781 mother-child dyads with children between the age group of 2 and 6 years. METHODS: Selected mothers were asked to complete the questionnaires on sociodemographic data, mother's oral health knowledge (MOHK), tooth-brushing practices, and MTBSE. Brushing adherence was evaluated as complete adherence if the child followed twice daily assisted brushing using the toothbrush and toothpaste. STATISTICAL ANALYSIS: Nonparametric tests were used to compare the variables. Binary logistic regression was used to evaluate the predictors of brushing adherence. RESULTS: Complete brushing adherence (assisted brushing with toothbrush and toothpaste at least twice per day) was seen only in 26.9% children. More children with complete brushing adherence were single children (P < 0.001). Children with complete brushing adherence had mothers with significantly higher MTBSE (P < 0.001). The presence/absence of siblings, MOHK, and MTBSE were found to be strong and significant predictors of brushing adherence in children. CONCLUSIONS: MTBSE plays a significant role in complete adherence to toothbrushing in children aged 2-6 years.

Colorectal Cancer-Associated Smad4 R361 Hotspot Mutations Boost Wnt/ß-Catenin Signaling through Enhanced Smad4-LEF1 Binding.

About 10% to 30% of patients with colorectal cancer harbor either loss of or missense mutations in SMAD4, a critical component of the TGFß signaling pathway. The pathophysiologic function of missense mutations in Smad4 is not fully understood. They usually map to the MH2 domain, specifically to residues that are involved in heterodimeric complex formation with regulatory Smads (such as Smad2/3) and ensuing transcriptional activation. These detrimental effects suggest that SMAD4 missense mutations can be categorized as loss-of-function. However, they tend to cluster in a few hotspots, which is more consistent with them acting by a gain-of-function mechanism. In this study, we investigated the functional role of Smad4 R361 mutants by re-expressing two R361 Smad4 variants in several Smad4-null colorectal cancer cell lines. As predicted, R361 mutations disrupted Smad2/3-Smad4 heteromeric complex formation and abolished canonical TGFß signaling. In that, they were similar to SMAD4 loss. However, RNA sequencing and subsequent RT-PCR assays revealed that Smad4mut cells acquired a gene signature associated with enhanced Lef1 protein function and increased Wnt signaling. Mechanistically, Smad4 mutant proteins retained binding to Lef1 protein and drove a commensurate increase in downstream Wnt signaling as measured by TOP/FOP luciferase assay and Wnt-dependent cell motility. Consistent with these findings, human colorectal cancers with SMAD4 missense mutations were less likely to acquire activating mutations in the key Wnt pathway gene CTNNB1 (encoding ß-catenin) than colorectal cancers with truncating SMAD4 nonsense mutations. IMPLICATIONS: Our studies suggest that in colorectal cancer hotspot mutations in Smad4 confer enhanced Wnt signaling and possibly heightened sensitivity to Wnt pathway inhibitors. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/5/823/F1.large.jpg.

Tissue-specific quantification and localization of androgen and estrogen receptors in prostate cancer.

Androgens and estrogens, working together, promote prostate cancer (PRCA) initiation and progression, with androgens acting via androgen receptor (AR) and estrogens acting primarily through estrogen receptor α (ERα). While the interplay between these steroid hormones has been established, the interaction between steroid hormone receptors in prostatic disease remains unstudied. The goal of this study was to objectively determine the incidence, stage specificity, and tissue/cell type specificity of AR and ERα expression, both independently and simultaneously, during the progression of PRCA. Using multiplexed immunohistochemistry and multispectral imaging analysis, AR, ERα, and smooth muscle α-actin expression was detected and quantitated in benign prostate tissue (BPT), high-grade prostatic intraepithelial neoplasia (HGPIN), PRCA, and metastasis (MET) from patient specimens (n=340). Epithelial AR expression was significantly increased in HGPIN, PRCA, and MET compared with BPT, whereas ERα expression in epithelial and stromal cells was highest in HGPIN. With analysis of AR and ERα coexpression, we identified a unique population of double-positive (AR+/ERα+) cells that increased in HGPIN specimens in both the stroma and the epithelium. Double-negative (AR-/ERα-) cells significantly decreased across PRCA progression, from 65% in BPT to 30% in MET. Preliminary analysis of this AR+/ERα+ population indicates potential cell type specificity in smooth muscle α-actin-negative stromal cells. This study demonstrates stage-, tissue-, and cell type-specific AR and ERα expression changes during PRCA progression, both independently and coexpressed. A more complete understanding of steroid hormones and their receptors in the initiation and progression of prostatic disease may elucidate improved strategies for PRCA prevention or therapy.

Does Combination Therapy with Tamsulosin and Tolterodine Improve Ureteral Stent Discomfort Compared with Tamsulosin Alone? A Double-Blind, Randomized, Controlled Trial.

PURPOSE: Ureteral stent discomfort is a significant postoperative problem for many patients. Despite the use of narcotics and α-blockers patients often experience bothersome lower urinary tract symptoms and pain, which impair daily activities. We compared combination therapy with an α-blocker and an anticholinergic to monotherapy with an α-blocker. MATERIALS AND METHODS: A double-blind, randomized, controlled trial was performed from December 2012 to April 2014. A total of 80 patients were randomized, including 44 to the combination group (tamsulosin 0.4 mg and tolterodine early release 4 mg) and 36 to the monotherapy group (tamsulosin 0.4 mg and placebo). Patients with preexisting ureteral stent placement or current anticholinergic therapy were excluded from study. Patients completed USSQ (Urinary Stent Symptom Questionnaire) before stent placement on the day of surgery, the day after stent placement, the morning of stent removal and the day after stent removal. The questionnaire included questions regarding urinary symptoms, general health, body pain, and work and sexual history. RESULTS: A total of 80 patients (40 males and 40 females) were studied. Mean age was 51.5 vs 51.3 years (p = 0.95) and mean body mass index was 33.6 vs 31.9 kg/m(2) (p = 0.44) in monotherapy group 1 vs combination therapy group 2. Between the 2 groups there was no significant difference in urinary symptoms, body pain and activities of daily living from baseline to just before stent removal (p = 0.95, 0.40 and 0.95, respectively). Although there was no difference between the groups, both showed improvement in urinary symptoms from the time of initial stent insertion to just prior to stent removal (difference -0.50 for combination therapy and -0.40 for monotherapy). The mean stent indwelling time of 9.6 and 8.7 days in the combination and monotherapy groups, respectively, did not differ (p = 0.67). On ANOVA it had no significant impact on results (p = 0.64). CONCLUSIONS: Combination therapy with tamsulosin and tolterodine does not appear to improve urinary symptoms, bodily pain or quality of life in patients after ureteral stent placement for nephrolithiasis compared to tamsulosin alone. Both groups experienced worse urinary symptoms, pain and quality of life with a stent, suggesting that further research is necessary to improve stent discomfort.

Soluble CD109 binds TGF-ß and antagonizes TGF-ß signalling and responses.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine implicated in many diseases, including tissue fibrosis and cancer. TGF-ß mediates diverse biological responses by signalling through type I and II TGF-ß receptors (TßRI and TßRII). We have previously identified CD109, a glycosylphosphatidylinositol (GPI)-anchored protein, as a novel TGF-ß co-receptor that negatively regulates TGF-ß signalling and responses and demonstrated that membrane-anchored CD109 promotes TGF-ß receptor degradation via a SMAD7/Smurf2-mediated mechanism. To determine whether CD109 released from the cell surface (soluble CD109 or sCD109) also acts as a TGF-ß antagonist, we determined the efficacy of recombinant sCD109 to interact with TGF-ß and inhibit TGF-ß signalling and responses. Our results demonstrate that sCD109 binds TGF-ß with high affinity as determined by surface plasmon resonance (SPR) and cell-based radioligand binding and affinity labelling competition assays. SPR detected slow dissociation kinetics between sCD109 and TGF-ß at low concentrations, indicating a stable and effective interaction. In addition, sCD109 antagonizes TGF-ß-induced Smad2/3 phosphorylation, transcription and cell migration. Together, our results suggest that sCD109 can bind TGF-ß, inhibit TGF-ß binding to its receptors and decrease TGF-ß signalling and TGF-ß-induced cellular responses.

SIGIRR/TIR8, an important regulator of TLR4 and IL-1R-mediated NF-κB activation, predicts biochemical recurrence after prostatectomy in low-grade prostate carcinomas.

Single Ig IL-1-related receptor (SIGIRR) is a negative regulator of toll-like receptor 4 and IL-1-mediated activation of nuclear factor κ-light-chain enhancer of activated B cells. The purpose of this study was to qualitatively and quantitatively determine SIGIRR protein expression in human prostate tissues and associate SIGIRR expression with clinical parameters. SIGIRR expression was quantified in glandular prostate tissue using immunohistochemistry and multispectral imaging, and expression was evaluated in relation to clinicopathological features of benign prostatic hyperplasia and prostate cancer (PCa). Subgroupings of low Gleason score (≤ 6 and 3 + 4) and high Gleason score (4 + 3 and ≥ 8) were used for patient outcomes. SIGIRR was predominantly expressed in the cytoplasm and nucleus of the prostatic epithelium with little expression within the stroma. Compared with normal prostate, cytoplasmic SIGIRR expression was similar in benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, PCa, and metastases. A decrease in nuclear expression was found in metastasis samples (P = .04). Changes in SIGIRR expression were not associated with Gleason score, pathological stage, tumor volume, surgical margin status, or serum prostate-specific antigen (P > .05). Nuclear (P = .96) and cytoplasmic (P = .89) SIGIRR expressions were not related to patient outcomes in univariable analysis, but in the analysis of patients with low Gleason scores, high cytoplasmic SIGIRR expression was associated with biochemical recurrence in both univariable (P = .01) and multivariable (hazard ratio, 2.31 [95% confidence interval 1.05-5.06]; P = .04) analyses. Similarly, in multivariable analysis of only low-stage (pT2) tumors, SIGIRR independently predicted biochemical recurrence (P = .009). We conclude that SIGIRR predicts biochemical recurrence in patients with low Gleason score and low pathological stage PCa.

Factors affecting outcomes of percutaneous nephrolithotomy in horseshoe kidneys.

OBJECTIVE: To analyze the patient- and procedure-related factors affecting the outcomes of percutaneous nephrolithotomy (PNL) in horseshoe kidneys (HSKs). METHODS: A retrospective analysis was done of patients with stones in HSKs treated with PNL in 3 referral centers between 1998 and 2013. Demographics, along with perioperative characteristics, were evaluated in detail as to whether or not they had an effect on the success and complication rates. RESULTS: A total of 54 HSKs with calculi in 53 patients were treated with PNL. Mean stone size was 28.4 ± 19.6 mm (range, 10-120 mm). Fifty-three patients were treated through a single tract, and 1 patient required additional access. Access was directed to the upper calyx (n = 27), middle calyx (n = 17), and lower calyx (n = 10) through the intercostal (n = 23) and subcostal (n = 31) areas. Flexible nephroscopy was used in 18.5% of the procedures. Postoperative complications were observed in 9 (16.7%) of the procedures. Success rate was 66.7% after a single session of PNL and increased to 90.7% with additional treatments. Although patient demographics, preoperative imaging, and other operative measures did not have significant effect on the complication rate, stone complexity and multiplicity, in combination with flexible nephroscopy, were found to significantly affect the success rate (P = .026, P = .043, and P = .021, respectively). However, in multivariate analysis stone multiplicity was the only factor that affected success rate (P = .004). CONCLUSION: Stone parameters play an important role in achieving stone-free status in HSKs. Use of flexible nephroscopy positively affects the success rate by allowing reaching the peripherally located calices.

Finasteride treatment alters tissue specific androgen receptor expression in prostate tissues.

BACKGROUND: Normal and pathologic growth of the prostate is dependent on the synthesis of dihydrotestosterone (DHT) from testosterone by 5α-reductase. Finasteride is a selective inhibitor of 5α-reductase 2, one isozyme of 5α-reductase found in abundance in the human prostate. The objective of this study was to investigate the effects of finasteride on androgen receptor expression and tissue morphology in human benign prostatic hyperplasia specimens. METHODS: Patients undergoing transurethral resection of the prostate and either treated or not treated with finasteride between 2004 and 2010 at the University of Wisconsin-Hospital were retrospectively identified using an institutional database. Prostate specimens from each patient were triple-stained for androgen receptor, prostate-specific antigen, and basal marker cytokeratin 5. Morphometric analysis was performed using the multispectral imaging, and results were compared between groups of finasteride treated and non-treated patients. RESULTS: Epithelial androgen receptor but not stromal androgen receptor expression was significantly lower in patients treated with finasteride than in non-treated patients. Androgen receptor-regulated prostate-specific antigen was not significantly decreased in finasteride-treated patients. Significant luminal epithelial atrophy and basal cell hyperplasia were prevalent in finasteride treated patients. Epithelial androgen receptor expression was highly correlated to the level of luminal epithelial atrophy. CONCLUSIONS: In this study, finasteride decreased the expression of epithelial androgen receptor in a tissue specific manner. The correlation between epithelial androgen receptor and the extent of luminal epithelial atrophy suggests that epithelial androgen receptor may be directly regulating the atrophic effects observed with finasteride treatment.

Expanding endourology for biliary stone disease: the efficacy of intracorporeal lithotripsy on refractory biliary calculi.

BACKGROUND AND PURPOSE: We evaluated the efficacy of ureteroscopic therapy (electrohydraulic lithotripsy [EHL] and intraductal laser lithotripsy [ILL]) in patients with challenging biliary stones secondary to anatomic variations resulting from a previous surgical procedure, including liver transplantation. PATIENTS AND METHODS: A retrospective chart review was performed for all patients with previous surgical alteration of the gastrointestinal (GI) tract who underwent EHL or ILL via peroral or percutaneous access for choledocholithiasis by a single surgeon at our institution from 2000 to 2012. A database containing clinical and surgical variables was created, and long-term follow-up was conducted (3-138 months; median, 99 months). RESULTS: Thirteen patients (51.7±20.0 years; M:F, 10:3) in whom endoscopic retrograde cholangiopancreatography (ERCP), percutaneous transhepatic cholangiography (PTHC), or both failed were identified. Failure of ERCP/PTHC was because of inaccessibility of the calculi in all cases. Stone clearance was achieved in 12/13 (93%) patients; 8/12 (62%) after one procedure, and 4/12 (31%) after two procedures. One patient with biliary cast syndrome needed four interventions over 9 years. Major complications were low, with only one patient with hypotension and cholangitis that resolved with 24 hours of administration of intravenous fluids and antibiotics. CONCLUSIONS: Both endoscopic and percutaneous lithotripsies are effective treatments for refractory biliary calculi resulting from the post-surgical GI tract. Although a staged second procedure may be necessary in patients with significant stone burden, this is significantly better than extensive open surgery.

Insulin like growth factor binding protein 4 promotes GBM progression and regulates key factors involved in EMT and invasion.

Insulin like growth factor binding protein 4 (IGFBP4) regulates growth and development of tissues and organs by negatively regulating IGF signaling. Among most cancers, IGFBP4 has growth inhibitory role and reported as a down-regulated gene, except for renal cell carcinoma, wherein IGFBP4 promotes tumor progression. IGFBP4 expression has been shown to be higher in increasing grades of astrocytoma. However, the functional role of IGFBP4 in gliomas has not been explored. Surgical biopsies of 20 normal brain and 198 astrocytoma samples were analyzed for IGFBP4 expression by qRT-PCR. Highest expression of IGFBP4 mRNA was seen in GBM tumors compared to control brain tissues (median log2 of 2.035, p < 0.0001). Immunohistochemical analysis of 53 tissue samples revealed predominant nuclear staining of IGFBP4, seen maximally in GBMs when compared to DA and AA tumors (median LI = 29.12 ± 16.943, p < 0.001). Over expression of IGFBP4 in U343 glioma cells resulted in up-regulation of molecules involved in tumor growth, EMT and invasion such as pAkt, pErk, Vimentin, and N-cadherin and down-regulation of E-cadherin. Functionally, IGFBP4 over expression in these cells resulted in increased proliferation, migration and invasion as assessed by MTT, transwell migration, and Matrigel invasion assays. These findings were confirmed upon IGFBP4 knockdown in U251 glioma cells. Our data suggest a pro-tumorigenic role for IGFBP4 in glioma.

Dietary hydroxyproline induced calcium oxalate lithiasis and associated renal injury in the porcine model.

BACKGROUND AND PURPOSE: We previously reported hyperoxaluria and calcium oxalate calculi in adult pigs (sows) fed hydroxyproline (HP). The purpose of this study was to grossly and histopathologically characterize intrarenal effects in this model. METHODS: In the swine facility at our campus, we maintained 21 gestating sows, of which 15 received daily treatment (5% HP mixed with dry feed) and 6 received no treatment (controls). Nine were sacrificed at 21 d (three control, six HP). All kidneys were extracted and examined grossly and for radiographic evidence of stones (GE CT scanner, 80kV, 400MA, 1 sec rotation, 0.625 mm slices). Papillary and cortical samples were processed for histologic analysis. RESULTS: Kidneys from treated sows showed significant calculi distributed within the renal papilla on CT, appeared mottled in the renal cortex and papillary areas, and had less distinct corticomedullary borders. Tiny crystals and mucinous debris lined the papillary tips, calices, and pelvis in kidneys from four of six treated sows, and multiple stones were noted at the papillary tips. Hematoxylin and eosin stain revealed crystals in collecting tubules and papillary tips in treated kidneys and none in controls. Yasue staining confirmed crystals in proximal periglomerular tubules of treated but not control animals. Tubular dilation and inflammatory/fibrotic changes were identified in kidneys from treated animals; none of these changes were evident in control kidneys. CONCLUSIONS: We report renal damage as a result of dietary-induced hyperoxaluria in adult sows. Specifically, we found crystalluria in proximal periglomerular tubules and collecting ducts, with tubular damage at all segments.

Regulation of protumorigenic pathways by insulin like growth factor binding protein2 and its association along with ß-catenin in breast cancer lymph node metastasis.

BACKGROUND: Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. METHODS: Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of ß-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for ß-catenin and IGFBP2 expression. RESULTS: Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of ß- catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of ß-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and ß-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. CONCLUSION: This study highlights regulation of ß-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and ß-catenin is associated with lymph node metastasis of breast tumors.

Sex steroid receptor expression and localization in benign prostatic hyperplasia varies with tissue compartment.

Androgens and estrogens, acting via their respective receptors, are important in benign prostatic hyperplasia (BPH). The goals of this study were to quantitatively characterize the tissue distribution and staining intensity of androgen receptor (AR) and estrogen receptor-alpha (ERα), and assess cells expressing both AR and ERα, in human BPH compared to normal prostate. A tissue microarray composed of normal prostate and BPH tissue was used and multiplexed immunohistochemistry was performed to detect AR and ERα. We used a multispectral imaging platform for automated scanning, tissue and cell segmentation and marker quantification. BPH specimens had an increased number of epithelial and stromal cells and increased percentage of epithelium. In both stroma and epithelium, the mean nuclear area was decreased in BPH relative to normal prostate. AR expression and staining intensity in epithelial and stromal cells was significantly increased in BPH compared to normal prostate. ERα expression was increased in BPH epithelium. However, stromal ERα expression and staining intensity was decreased in BPH compared to normal prostate. Double positive (AR and ERα) epithelial cells were more prevalent in BPH, and fewer double negative (AR and ERα) stromal and epithelial negative cells were observed in BPH. These data underscore the importance of tissue layer localization and expression of steroid hormone receptors in the prostate. Understanding the tissue-specific hormone action of androgens and estrogens will lead to a better understanding of mechanisms of pathogenesis in the prostate and may lead to better treatment for BPH.

Microfluidic encapsulation of cells in alginate capsules for high throughput screening.

Microdroplet systems can drastically reduce costs and increase throughput in high throughput screening (HTS) assays. While droplets are well suited for biomolecular screening, cell-based screens are more problematic because eukaryotes typically require attachment to solid supports to maintain viability and function. This paper describes an economical, off-the-shelf microfluidic system which encapsulates eukaryotic cells in gelatinous alginate capsules for the purpose of HTS. The flow-through system consists of i) a cross junction, which forms monodisperse droplets of alginate and cell suspension in an immiscible carrier fluid, followed by ii) a T junction which delivers BaCl(2) to crosslink and solidify each droplet. With an appropriate carrier fluid, the system is self-synchronized and can produce cell-alginate-BaCl(2) capsules with virtually 100% reliability. Droplet volumes and frequency are determined by flow rates and the diameter of the cross junction. The present implementation, which utilizes 1.5 mm Teflon tubing and plastic junctions, can generate 0.4-1.4 microL droplets at frequencies >10 droplets/sec. Cell viability is >80% at 4 hours post-encapsulation. With low recurring cost (