Neurofilament Light Chain as a Biomarker of Global Cognition in Individuals With Possible Vascular Mild Cognitive Impairment.

BACKGROUND: Neurofilament Light Chain (NfL) is a biomarker of axonal injury elevated in mild cognitive impairment (MCI) and Alzheimer's disease dementia. Blood NfL also inversely correlates with cognitive performance in those conditions. However, few studies have assessed NfL as a biomarker of global cognition in individuals demonstrating mild cognitive deficits who are at risk for vascular-related cognitive decline. OBJECTIVE: To assess the relationship between blood NfL and global cognition in individuals with possible vascular MCI (vMCI) throughout cardiac rehabilitation (CR). Additionally, NfL levels were compared to age/sex-matched cognitively unimpaired (CU) controls. METHOD: Participants with coronary artery disease (vMCI or CU) were recruited at entry to a 24-week CR program. Global cognition was measured using the Montreal Cognitive Assessment (MoCA) and plasma NfL level (pg/ml) was quantified using a highly sensitive enzyme-linked immunosorbent assay. RESULTS: Higher plasma NfL was correlated with worse MoCA scores at baseline (ß = -.352, P = .029) in 43 individuals with vMCI after adjusting for age, sex, and education. An increase in NfL was associated with worse global cognition (b[SE] = -4.81[2.06], P = .023) over time, however baseline NfL did not predict a decline in global cognition. NfL levels did not differ between the vMCI (n = 39) and CU (n = 39) groups (F(1, 76) = 1.37, P = .245). CONCLUSION: Plasma NfL correlates with global cognition at baseline in individuals with vMCI, and is associated with decline in global cognition during CR. Our findings increase understanding of NfL and neurobiological mechanisms associated with cognitive decline in vMCI.

Mesonephric-like adenocarcinoma of the female genital tract: novel observations and detailed molecular characterisation of mixed tumours and mesonephric-like carcinosarcomas.

AIMS: To report novel observations in five mesonephric-like adenocarcinomas (MLAs) of the female genital tract. METHODS AND RESULTS: We report two endometrial MLAs in association with endometrioid carcinoma and atypical hyperplasia and three (one endometrial, two ovarian) cases with a sarcomatoid component (mesonephric-like carcinosarcoma). Pathogenic KRAS mutations, which are characteristic of MLA, were identified in all cases although interestingly, in one of the mixed carcinomas, this was confined to the endometrioid component. The concurrent MLA, endometrioid carcinoma and atypical hyperplasia components in one case harboured identical EGFR, PTEN and CCNE1 mutations, suggesting that the atypical hyperplasia gave rise to a Müllerian carcinoma with both endometrioid and mesonephric-like components. The carcinosarcomas all contained a component of MLA and a sarcomatous component with chondroid elements. In the ovarian carcinosarcomas, the coexisting epithelial and sarcomatous components shared some mutations including KRAS and CREBBP, suggesting that they are clonally related. Furthermore, in one case CREBBP and KRAS mutations detected in the MLA and sarcomatous components were also detected in an associated undifferentiated carcinoma component, suggesting that it was clonally related to the MLA and sarcomatous components. CONCLUSIONS: Our observations provide additional evidence that MLAs have a Müllerian origin and characterise mesonephric-like carcinosarcomas in which chondroid elements appear to be characteristic. In reporting these findings, we provide recommendations for distinction between a mesonephric-like carcinosarcoma and a MLA with a spindle cell component.

Genomic profiling of dedifferentiated endometrial carcinomas arising in the background of high-grade carcinoma: a targeted next-generation sequencing study.

AIMS: Our understanding of dedifferentiated endometrial carcinoma (DEC), a rare and aggressive malignancy, mainly reflects undifferentiated carcinomas (UC) arising in the setting of low-grade endometrial cancer (DEC-LG). However, cases of UC arising in the setting of high-grade EC (DEC-HG) have been noted in the literature. Our knowledge of the genomics of DEC-HG is limited. To characterise the molecular landscape of DEC-HC, targeted genomic sequencing and immunohistochemical analysis was carried out on seven DEC-HG and four DEC-LG. METHODS AND RESULTS: DEC-HG and DEC-LG, including undifferentiated and differentiated components, both showed a similar frequency and spectrum of mutations. ARID1A mutations were identified in 6/7 (86%) DEC-HG and 4/4 (100%) DEC-LG, while SMARCA4 mutations were present in 4/7 (57%) DEC-HG and in 1/4 (25%) DEC-LG. Concurrent SMARCA4/BRG1 protein loss by immunohistochemistry was observed in 3/4 and 1/1 SMARCA4 mutated DEC-HG and DEC-LG, respectively. Neither genomic alterations nor protein loss in SMARCB1/INI1 were observed in any of our cases. TP53 mutations were detected in 4/7 (57%) DEC-HG and in 2/4 (50%) DEC-LG, while mutation-pattern p53 immunohistochemistry expression was observed in 2/7 (29%) DEC-HG and none of the DEC-LG. MLH1 mutations were observed in 1/7 (14%) DEC-HG and 1/4 (25%) DEC-LG. MSH2 and MSH6 mutations were each detected in 1/7 (14%) DEC-HG, but neither was associated with corresponding loss of protein expression. CONCLUSION: The findings support expanding the definition of DEC to include DEC-HG, a previously under-recognised phenomenon with genomic similarities to DEC-LG.

Differentiated exophytic vulvar intraepithelial lesion: Clinicopathologic and molecular analysis documenting its relationship with verrucous carcinoma of the vulva.

Verruciform proliferations of the vulva unrelated to HPV infection are rare. The term differentiated exophytic vulvar intraepithelial lesion (DEVIL) was recently proposed for these lesions, which harbor recurrent PIK3CA mutations. It is still unclear whether DEVIL is related to verrucous carcinoma, a neoplasm characterized by persistence and local recurrence but nil risk of distant spread. Specimens identified using the words "verruciform" and "verrucous" were reviewed. Diagnosis of DEVIL required verruciform acanthosis, hyper and/or parakeratosis, hypogranulosis, cytoplasmic pallor, and bland nuclei. Verrucous carcinoma required, in addition, discontinuous, bulbous, puzzle-like nests in the stroma. A targeted next-generation sequencing using a custom 11-gene panel was performed. Eighteen specimens corresponding to ten patients with DEVIL and/or verrucous carcinoma were included. Median age at presentation was 66 years for DEVIL and 70 years for verrucous carcinoma. A similar spectrum of prevalent mutations was found in both lesions involving HRAS, PIK3CA, and BRAF. DEVIL preceded verrucous carcinoma and/or was diagnosed concurrently or in subsequent follow-up in five patients. In four of these, the same mutation was identified in DEVIL and synchronous or metachronous carcinoma. All cases showed wild-type 53 staining and lacked pathogenic TP53 mutations. DEVIL is a rare form of squamous proliferation characterized by prevalent PIK3CA and HRAS mutations. Its temporal relationship with verrucous carcinoma and their shared mutational profile in some patients suggest that DEVIL is a precursor of verrucous carcinoma. Moreover, given their morphologic and molecular overlap and the nil risk of verrucous carcinoma for distant spread, it is conceivable that DEVIL and verrucous carcinoma represent a spectrum of the same entity.

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern, North African, and South European countries.

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population-specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.

MicroRNA-139 is a predictor of prostate cancer recurrence and inhibits growth and migration of prostate cancer cells through cell cycle arrest and targeting IGF1R and AXL.

BACKGROUND: We previously identified a panel of five microRNAs (miRNAs) associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using next-generation sequencing-based whole miRNome sequencing and quantitative polymerase chain reaction-based validation analysis. In this study, we examined the mechanism of action of miR-139-5p, one of the downregulated miRNAs identified in the panel. METHODS: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan-Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that overexpressed miR-139 to confirm its targets as well as examine pathways through which miR-139 may function using cell-based assays. RESULTS: Low miR-139 expression was significantly associated with a variety of prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity, and lymph node status. MiR-139 expression was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis were significantly lower among patients with high miR-139 expression (P = .0004 and .038, respectively). Validation in the TCGA data set showed a significant association between dichotomized miR-139 expression and biochemical recurrence (odds ratio, 0.52; 95% confidence interval, 0.33-0.82). Overexpression of miR-139 in prostate cancer cells led to a significant reduction in cell proliferation and migration compared with control cells, with cells arrested in G2 of cell cycle. IGF1R and AXL were identified as potential targets of miR-139 based on multiple miRNA-binding sites in 3'-untranslated regions of both the genes and their association with prostate cancer growth pathways. Luciferase assays verified AXL and IGF1R as direct targets of miR-139. Furthermore, immunoblotting of prostate cancer cells demonstrated IGF1R and AXL protein expression were inhibited by miR-139 treatment, which was reversed by the addition of miR-139 antagomir. Examination of the molecular mechanism of growth inhibition by miR-139 revealed the downregulation of activated AKT and cyclin D1, with upregulation of the CDK inhibitor p21. CONCLUSIONS: miR-139 is associated with improved prognosis in patients with localized prostate cancer, which may be mediated through downregulation of IGF1R and/or AXL and associated signaling pathway components.

Overview of transcriptomic analysis of all human proteases, non-proteolytic homologs and inhibitors: Organ, tissue and ovarian cancer cell line expression profiling of the human protease degradome by the CLIP-CHIP™ DNA microarray.

The protease degradome is defined as the complete repertoire of proteases and inhibitors, and their nonfunctional homologs present in a cell, tissue or organism at any given time. We review the tissue distribution of virtually the entire degradome in 23 different human tissues and 6 ovarian cancer cell lines. To do so, we developed the CLIP-CHIP™, a custom microarray based on a 70-mer oligonucleotide platform, to specifically profile the transcripts of the entire repertoire of 473 active human proteases, 156 protease inhibitors and 92 non-proteolytically active homologs known at the design date using one specific 70-mer oligonucleotide per transcript. Using the CLIP-CHIP™ we mapped the expression profile of proteases and their inhibitors in 23 different human tissues and 6 ovarian cancer cell lines in 104 sample datasets. Hierarchical cluster analysis showed that expression profiles clustered according to their anatomic locations, cellular composition, physiologic functions, and the germ layer from which they are derived. The human ovarian cancer cell lines cluster according to malignant grade. 110 proteases and 42 inhibitors were tissue specific (1 to 3 tissues). Of these 110 proteases 69% (74) are mainly extracellular, 30% (34) intracellular and 1% intramembrane. Notably, 35% (197/565) of human proteases and 30% (47/156) of inhibitors were ubiquitously expressed in all 23 tissues; 27% (155) of proteases and 21% (32) of inhibitors were broadly expressed in 4-20 tissues. Our datasets provide a valuable resource for the community of baseline protease and inhibitor relative expression in normal human tissues and can be used for comparison with diseased tissue, e.g. ovarian cancer, to decipher pathogenesis, and to aid drug development. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Genomic abnormalities in invasive endocervical adenocarcinoma correlate with pattern of invasion: biologic and clinical implications.

The pattern-based classification system for HPV-related endocervical adenocarcinoma, which classifies tumors based on the destructiveness of stromal invasion, is predictive of the risk of nodal metastases and adverse outcome. Previous studies have demonstrated clinically important molecular alterations in endocervical adenocarcinoma, including KRAS and PIK3CA mutations; however, correlation between the molecular landscape and pathological variables including pattern of invasion has not been thoroughly explored. In this study, 20 endocervical adenocarcinomas were classified using the pattern-based classification system and were subjected to targeted sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 (ThermoFisher Scientific, Waltham, MA, USA) that surveys hotspot regions of 50 oncogenes and tumor suppressor genes. Single-nucleotide polymorphisms were correlated with clinical and pathologic variables including pattern of invasion. Five (25%), six (30%), and nine (45%) cases were classified as patterns A, B, and C respectively. Lymph node metastases, advanced stage at presentation and mortality from disease were exclusively seen in destructively invasive tumors (patterns B or C). Prevalent mutations in the cohort involved PIK3CA (30%), KRAS (30%), MET (15%), and RB1 (10%). Most (94%) relevant genomic alterations were present in destructively invasive tumors with PIK3CA, KRAS, and RB1 mutations seen exclusively in pattern B or C subgroups. KRAS mutations correlated with advanced stage at presentation (FIGO stage II or higher). Our findings indicate that the pattern of stromal invasion correlates with genomic abnormalities detected by next-generation sequencing, suggesting that tumors without destructive growth (pattern A) are biologically distinct from those with destructive invasion (patterns B and C), and that pattern B endocervical adenocarcinoma is more closely related to its pattern C counterpart. The pattern-based classification may be used as a triage tool when considering molecular testing for prognostic or therapeutic purposes.

Estimating the effect of immortal-time bias in urological research: a case example of testosterone-replacement therapy.

OBJECTIVE: To quantify the effect of immortal-time bias in an observational study examining the effect of cumulative testosterone exposure on mortality. PATIENTS AND METHODS: We used a population-based matched cohort study of men aged ≥66 years, newly treated with testosterone-replacement therapy (TRT), and matched-controls from 2007 to 2012 in Ontario, Canada to quantify the effects of immortal-time bias. We used generalised estimating equations to determine the association between cumulative TRT exposure and mortality. Results produced by models using time-fixed and time-varying exposures were compared. Further, we undertook a systematic review of PubMed to identify studies addressing immortal-time bias or time-varying exposures in the urological literature and qualitatively summated these. RESULTS: Among 10 311 TRT-exposed men and 28 029 controls, the use of a time-varying exposure resulted in the attenuation of treatment effects compared with an analysis that did not account for immortal-time bias. While both analyses showed a decreased risk of death for patients in the highest tertile of TRT exposure, the effect was overestimated when using a time-fixed analysis (adjusted hazard ratio [aHR] 0.56, 95% confidence interval [CI]: 0.52-0.61) when compared to a time-varying analysis (aHR 0.67, 95% CI: 0.62-0.73). Of the 1 241 studies employing survival analysis identified in the literature, nine manuscripts met criteria for inclusion. Of these, five used a time-varying analytical method. Each of these was a large, population-based retrospective cohort study assessing potential harms of pharmacological agents. CONCLUSIONS: Where exposures vary over time, a time-varying exposure is necessary to draw meaningful conclusions. Failure to use a time-varying analysis will result in overestimation of a beneficial effect. However, time-varying exposures are uncommonly utilised among manuscripts published in prominent urological journals.

MiR-301a regulates E-cadherin expression and is predictive of prostate cancer recurrence.

BACKGROUND: MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression post-transcriptionally. Dysregulation of miRNA has been implicated in the development and progression of prostate cancer. Through next generation miRNA sequencing, we recently identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Of the five miRNAs, miR-301a had the strongest association with prostate cancer recurrence. Overexpression of miR-301a in prostate cancer cells, PC3, and LNCaP resulted in increased growth both in vitro and in xenografted tumors. We therefore sought to examine its role in prostate carcinogenesis in greater detail. METHODS: We examined the effect of miR-301a expression on biochemical recurrence and metastasis among 585 men treated with radical prostatectomy for prostate cancer. We examined the mechanism of growth deregulation by miR-301a in prostate cancer cells using analysis of the miRome of prostate cancer cell lines, quantitative PCR, and Western blotting. RESULTS: High levels of miR-301a (above the median) were associated with an increased risk of biochemical recurrence (adjusted hazard ratio [aHR] 1.42, 95% confidence interval (CI) 1.06-1.90, P = 0.002) but not of metastasis (aHR 0.84, 95%CI 0.41-1.70, P = 0.6) after adjustment for known prognostic factors. RNA transcriptome sequencing analysis of miR-301a overexpressing prostate cancer cell lines identified the tumor suppressor p63 as a potential direct miR-301a target. Transcriptome sequencing, qPCR and Western blotting showed that miR-301a induced epithelial-mesenchymal transition (EMT) in prostate cancer cells through a pathway initiated by p63 inhibition. Luciferase assay verified p63 as a direct target of miR-301a. Loss of p63 resulted in miR-205 downregulation, releasing Zeb1 and Zeb2 from inhibition, culminating in Zeb1/Zeb2 suppression of E-cadherin. This pathway of growth alteration mediated by miR-301a upregulation was shown to be valid in prostate cancer cell lines and patient-derived tumors. CONCLUSIONS: These data indicate that miR-301a functions as an oncogene in prostate cancer by directly targeting the p63 tumor suppressor leading to loss of E-cadherin and EMT. Hence, miR-301a may serve as a novel biomarker in prostate cancer as well as a therapeutic target for prostate cancer management. Prostate 76:869-884, 2016. © 2016 Wiley Periodicals, Inc.

Increasing hospital admission rates for urological complications after transrectal ultrasound guided prostate biopsy.

PURPOSE: Transrectal ultrasound guided prostate biopsy is widely used to confirm the diagnosis of prostate cancer. The technique has been associated with significant morbidity in a small proportion of patients. MATERIALS AND METHODS: We conducted a population based study of 75,190 men who underwent a transrectal ultrasound guided biopsy in Ontario, Canada, between 1996 and 2005. We used hospital and cancer registry administrative databases to estimate the rates of hospital admission and mortality due to urological complications associated with the procedure. RESULTS: Of the 75,190 men who underwent transrectal ultrasound biopsy 33,508 (44.6%) were diagnosed with prostate cancer and 41,682 (55.4%) did not have prostate cancer. The hospital admission rate for urological complications within 30 days of the procedure for men without cancer was 1.9% (781/41,482). The 30-day hospital admission rate increased from 1.0% in 1996 to 4.1% in 2005 (p for trend <0.0001). The majority of hospital admissions (72%) were for infection related reasons. The probability of being admitted to hospital within 30 days of having the procedure increased 4-fold between 1996 and 2005 (OR 3.7, 95% CI 2.0-7.0, p <0.0001). The overall 30-day mortality rate was 0.09% but did not change during the study period. CONCLUSIONS: The hospital admission rates for complications following transrectal ultrasound guided prostate biopsy have increased dramatically during the last 10 years primarily due to an increasing rate of infection related complications.

BCA2 is differentially expressed in renal oncocytoma: an analysis of 158 renal neoplasms.

The distinction between renal oncocytoma and renal cell carcinoma, especially chromophobe renal cell carcinoma and clear cell carcinoma with oncocytic features, is important due to the different biologic potentials of these tumors. RING E3 ligases have the subject of intense studies for their roles in many diseases including cancer and as potential therapeutic targets. All RING E3 ligases, including BCA2, contain a consensus protein sequence that would complex two or more zinc ions in the expressed protein. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics in these tumors. The ubiquitin-proteasome system regulates the turnover of proteins that have essential roles in the cell cycle, apoptosis, DNA damage repair, and in protein trafficking, which makes this pathway a target for oncogenic events. In this study, we investigated expression of BCA2 in renal oncocytoma and renal cell carcinoma. A total of 158 patients were included in the study. Our study has shown that 114/114 (100 %) cases of renal cell carcinoma were negative for BCA2. All 38 (100 %) cases of renal oncocytoma were positive for BCA2, and 6/6 (100 %) cases designated as oncocytic neoplasm which favor oncocytoma were also positive for BCA2. This is the first study to date evaluating the expression of BCA2 in renal oncocytoma. BCA2 could serve as a marker that may be utilized in the distinction between renal oncocytoma and its mimickers.

Host Expression Profiling From Diagnostic Coronavirus Disease 2019 Swabs Associates Upper Respiratory Tract Immune Responses With Radiologic Lung Pathology and Clinical Severity.

Background: COVID-19 presents with a breadth of symptomatology including a spectrum of clinical severity requiring intensive care unit (ICU) admission. We investigated the mucosal host gene response at the time of gold standard COVID-19 diagnosis using clinical surplus RNA from upper respiratory tract swabs. Methods: Host response was evaluated by RNA-sequencing, and transcriptomic profiles of 44 unvaccinated patients including outpatients and in-patients with varying levels of oxygen supplementation were included. Additionally, chest X-rays were reviewed and scored for patients in each group. Results: Host transcriptomics revealed significant changes in the immune and inflammatory response. Patients destined for the ICU were distinguished by the significant upregulation of immune response pathways and inflammatory chemokines, including cxcl2 which has been linked to monocyte subsets associated with COVID-19 related lung damage. In order to temporally associate gene expression profiles in the upper respiratory tract at diagnosis of COVID-19 with lower respiratory tract sequalae, we correlated our findings with chest radiography scoring, showing nasopharygeal or mid-turbinate sampling can be a relevant surrogate for downstream COVID-19 pneumonia/ICU severity. Conclusions: This study demonstrates the potential and relevance for ongoing study of the mucosal site of infection of SARS-CoV-2 using a single sampling that remains standard of care in hospital settings. We highlight also the archival value of high quality clinical surplus specimens, especially with rapidly evolving COVID-19 variants and changing public health/vaccination measures.

IGFBP7 reduces breast tumor growth by induction of senescence and apoptosis pathways.

Insulin-like growth factor binding protein 7 (IGFBP7) has been shown to be a tumor suppressor in a variety of cancers. We previously have shown that IGFBP7 expression is inversely correlated with disease progression and poor outcome in breast cancer. Overexpression of IGFBP7 in MDA-MB-468, a triple-negative breast cancer (TNBC) cell line, resulted in inhibition of growth and migration. Xenografted tumors bearing ectopic IGFBP7 expression were significantly growth-impaired compared to IGFBP7-negative controls, which suggested that IGFBP7 treatment could inhibit breast cancer cell growth. To confirm this notion, 14 human patient primary breast tumors were analyzed by qRTPCR for IGFBP7 expression. The TNBC tumors expressed the lowest levels of IGFBP7 expression, which also correlated with higher tumorigenicity in mice. Furthermore, when breast cancer cell lines were treated with IGFBP7, only the TNBC cell lines were growth inhibited. Treatment of NOD/SCID mice harboring xenografts of TNBC cells with IGFBP7 systemically every 3-4 days inhibited tumorigenesis, with associated anti-angiogenic effects, together with increased apoptosis. Upon examining the mechanism of IGFBP7-mediated growth inhibition in TNBC cells, we found that cells not only were arrested in G1 phase of the cell cycle but also underwent senescence as a result of treatment with IGFBP7. Interestingly, IGFBP7 treatment was also associated with strong activation of the stress-associated p38 MAPK pathway, together with upregulation of p53 and the cyclin-dependent protein kinase (CDK) inhibitor, p21(cip1). Prolonged treatment of cells with IGFBP7 resulted in increased cell death, marked by an increase in apoptotic cells and associated cleaved PARP. This is the first study showing that exogenous IGFBP7 inhibits TNBC cell growth both in vitro and in vivo. Taken together, these results suggest IGFBP7 treatment might have therapeutic potential for TNBC.

Protein-coding and microRNA biomarkers of recurrence of prostate cancer following radical prostatectomy.

An important challenge in prostate cancer research is to develop effective predictors of tumor recurrence following surgery to determine whether immediate adjuvant therapy is warranted. To identify biomarkers predictive of biochemical recurrence, we isolated the RNA from 70 formalin-fixed, paraffin-embedded radical prostatectomy specimens with known long-term outcomes to perform DASL expression profiling with a custom panel that we designed of 522 prostate cancer-relevant genes. We identified a panel of 10 protein-coding genes and two miRNA genes (RAD23B, FBP1, TNFRSF1A, CCNG2, NOTCH3, ETV1, BID, SIM2, LETMD1, ANXA1, miR-519d, and miR-647) that could be used to separate patients with and without biochemical recurrence (P < 0.001), as well as for the subset of 42 Gleason score 7 patients (P < 0.001). We performed an independent validation analysis on 40 samples and found that the biomarker panel was also significant at prediction of biochemical recurrence for all cases (P = 0.013) and for a subset of 19 Gleason score 7 cases (P = 0.010), both of which were adjusted for relevant clinical information including T-stage, prostate-specific antigen, and Gleason score. Importantly, these biomarkers could significantly predict clinical recurrence for Gleason score 7 patients. These biomarkers may increase the accuracy of prognostication following radical prostatectomy using formalin-fixed specimens.

Integrin-dependent Akt1 activation regulates PGC-1 expression and fatty acid oxidation.

BACKGROUND: Poly-N-acetyl glucosamine nanofibers derived from a marine diatom have been used to increase cutaneous wound healing. These nanofibers exert their activity by specifically activating integrins, which makes them a useful tool for dissecting integrin-mediated pathways. We have shown that short-fiber poly-N-acetyl glucosamine nanofiber (sNAG) treatment of endothelial cells results in increased cell motility and metabolic rate in the absence of increased cell proliferation. RESULTS: Using a Seahorse Bioanalyzer to measure oxygen consumption in real time, we show that sNAG treatment increases oxygen consumption rates, correlated with an integrin-dependent activation of Akt1. Akt1 activation leads to an increase in the expression of the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). This is not due to increased mitochondrial biogenesis, but is associated with an increase in the expression of pyruvate dehydrogenase kinase 4 (PDK4), suggesting regulation of fatty acid oxidation. Blockade of fatty acid oxidation with etomoxir, an O-carnitine palmitoyltransferase-1 inhibitor, blocks the sNAG-dependent increased oxygen consumption. (3)H-palmitate uptake experiments indicate a PDK4-dependent increase in fatty acid oxidation, which is required for nanofiber-induced cell motility. CONCLUSIONS: Our findings imply a linear pathway whereby an integrin-dependent activation of Akt1 leads to increased PGC-1α and PDK4 expression resulting in increased energy production by fatty acid oxidation.

Effects of partner proteins on BCA2 RING ligase activity.

BACKGROUND: BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2. METHODS: Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays. RESULTS: Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways. CONCLUSIONS: The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.

The role of versican G3 domain in regulating breast cancer cell motility including effects on osteoblast cell growth and differentiation in vitro - evaluation towards understanding breast cancer cell bone metastasis.

BACKGROUND: Versican is detected in the interstitial tissues at the invasive margins of breast carcinoma, is predictive of relapse, and negatively impacts overall survival rates. The versican G3 domain is important in breast cancer cell growth, migration and bone metastasis. However, mechanistic studies evaluating versican G3 enhanced breast cancer bone metastasis are limited. METHODS: A versican G3 construct was exogenously expressed in the 66c14 and the MC3T3-E1 cell line. Cells were observed through light microscopy and viability analyzed by Coulter Counter or determined with colorimetric proliferation assays. The Annexin V-FITC apoptosis detection kit was used to detect apoptotic activity. Modified Chemotactic Boyden chamber migration invasion assays were applied to observe tumor migration and invasion to bone stromal cells and MC3T3-E1 cells. Alkaline phosphatase (ALP) staining and ALP ELISA assays were performed to observe ALP activity in MC3T3-E1 cells. RESULTS: In the four mouse breast cancer cell lines 67NR, 66c14, 4T07, and 4T1, 4T1 cells expressed higher levels of versican, and showed higher migration and invasion ability to MC3T3-E1 cells and primary bone stromal cells. 4T1 conditioned medium (CM) inhibited MC3T3-E1 cell growth, and even lead to apoptosis. Only 4T1 CM prevented MC3T3-E1 cell differentiation, noted by inhibition of alkaline phosphatase (ALP) activity. We exogenously expressed a versican G3 construct in a cell line that expresses low versican levels (66c14), and observed that the G3-expressing 66c14 cells showed enhanced cell migration and invasion to bone stromal and MC3T3-E1 cells. This observation was prevented by selective EGFR inhibitor AG1478, selective MEK inhibitor PD 98059, and selective AKT inhibitor Triciribine, but not by selective JNK inhibitor SP 600125. Versican G3 enhanced breast cancer cell invasion to bone stromal cells or osteoblast cells appears to occur through enhancing EGFR/ERK or AKT signaling. G3 expressing MC3T3-E1 cells showed inhibited cell growth and cell differentiation when cultured with TGF-ß1 (1 ng/ml), and expressed enhanced cell apoptosis when cultured with TNF-α (2 ng/ml). Enhanced EGFR/JNK signaling appears to be responsible for G3 enhanced osteoblast apoptosis and inhibited osteoblast differentiation. Whereas repressed expression of GSK-3ß (S9P) contributes to G3 inhibited osteoblast growth. Versican G3 functionality was dependent on its EGF-like motifs. Without the structure of EGF-like repeats, the G3 domain would not confer enhancement of tumor cell migration and invasion to bone with concordant inhibition of osteoblast differentiation and promotion of osteoblast apoptosis. CONCLUSIONS: Versican enhances breast cancer bone metastasis not only through enhancing tumor cell mobility, invasion, and survival in bone tissues, but also by inhibiting pre-osteoblast cell growth, differentiation, which supply favorable microenvironments for tumor metastasis.

PAXgene Fixation for Pancreatic Cancer: Implications for Molecular and Surgical Pathology.

Genomic profiling of pancreatic cancer using small core biopsies has taken an increasingly prominent role in precision medicine. However, if not appropriately preserved, nucleic acids (NA) from pancreatic tissues are known to be susceptible to degradation due to high intrinsic levels of nucleases. PAXgene fixation (PreAnalytix, Switzerland) represents a novel formalin-free tissue preservation method. We sought to compare the NA and histomorphological preservation of pancreatic cancer tissues preserved with PAXgene-fixed paraffin-embedding (PFPE) and formalin-fixed paraffin-embedding (FFPE). Tissues from 19 patients were obtained prospectively from pancreaticoduodenectomy specimens and evaluated by four gastrointestinal pathologists. The extracted NA were quantified by Nanodrop and Qubit and assessed for quality by qPCR, targeted next-generation sequencing (NGS) assay, and RNA-sequencing. Our results demonstrated that, when assessed blindly for morphological quality, the four pathologists deemed the PFPE slides adequate for diagnostic purposes. PFPE tissues enable greater yields of less fragmented and more amplifiable DNA. PFPE tissues demonstrated significantly improved quality control (QC) metrics in a targeted NGS assay including Median Absolute Pair-wise Difference (MAPD) scores. Our results support the use of PAXgene fixative for the processing of specimens from pancreatic cancers with the potential benefits of improved yields for more amplifiable DNA in low-yield biopsy specimens and its ideal use for amplicon-based NGS assays.

Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors.

Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein.