Hemangiosarcoma Cells Promote Conserved Host-Derived Hematopoietic Expansion.

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel-forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors.

Digital and manual interfollicular Ki-67 are associated with a progression-free survival in patients with low-grade follicular lymphoma.

OBJECTIVES: Novel histopathologic prognostic factors are needed to identify patients with follicular lymphoma (FL) at risk of inferior outcomes. Our primary objective was to evaluate the Ki-67 proliferative index in follicular and interfollicular areas in tissue biopsy specimens from patients with newly diagnosed FL and correlate with clinical outcomes. Our secondary objective was to correlate PD-L1 and LAG-3 with clinical outcomes. METHODS: Seventy cases of low-grade FL from the University of Minnesota were evaluated with Ki-67 immunohistochemical stain. Ki-67 expression as a continuous variable was interpreted digitally and manually in follicular and interfollicular areas. Progression-free survival (PFS) and overall survival (OS) were analyzed by Cox regression, and hazard ratios (HRs) per 10-point increase in Ki-67 were calculated. RESULTS: Progression-free survival at 4 years was 28% (95% CI, 19%-41%). Interfollicular, but not follicular, Ki-67 was associated with PFS by manual (HR, 1.33; P = .01) and digital (HR, 1.38; P = .02) analysis. Digital and manual Ki-67 were only moderately correlated but demonstrated similar effects on PFS. At 4 years, OS was 90% with no association with follicular or interfollicular Ki-67 proliferation. CONCLUSIONS: Higher interfollicular Ki-67 by either digital or manual analysis is associated with a poorer PFS in patients with low-grade FL. These results suggest further validation of this marker is warranted to improve pathologic risk stratification at FL diagnosis. PD-L1 and LAG-3 were not associated with PFS or OS.

Receptor for hyaluronan-mediated motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients.

Breast cancer invasion and metastasis result from a complex interplay between tumor cells and the tumor microenvironment (TME). Key oncogenic changes in the TME include aberrant synthesis, processing, and signaling of hyaluronan (HA). Hyaluronan-mediated motility receptor (RHAMM, CD168; HMMR) is an HA receptor enabling tumor cells to sense and respond to this aberrant TME during breast cancer progression. Previous studies have associated RHAMM expression with breast tumor progression; however, cause and effect mechanisms are incompletely established. Focused gene expression analysis of an internal breast cancer patient cohort confirmed that increased RHAMM expression correlates with aggressive clinicopathological features. To probe mechanisms, we developed a novel 27-gene RHAMM-related signature (RRS) by intersecting differentially expressed genes in lymph node (LN)-positive patient cases with the transcriptome of a RHAMM-dependent model of cell transformation, which we validated in an independent cohort. We demonstrate that the RRS predicts for poor survival and is enriched for cell cycle and TME-interaction pathways. Further analyses using CRISPR/Cas9-generated RHAMM-/- breast cancer cells provided direct evidence that RHAMM promotes invasion in vitro and in vivo. Immunohistochemistry studies highlighted heterogeneous RHAMM protein expression, and spatial transcriptomics associated the RRS with RHAMM-high microanatomic foci. We conclude that RHAMM upregulation leads to the formation of 'invasive niches', which are enriched in RRS-related pathways that drive invasion and could be targeted to limit invasive progression and improve patient outcomes. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Expression and Prognostic Evaluation of the Receptor Tyrosine Kinase MET in Canine Malignant Melanoma.

The overexpression and activation of the MET receptor tyrosine kinase has been identified in many human malignancies, but its role in canine cancer has only been minimally investigated. In this study we evaluated the expression of MET in two canine malignant melanoma (CMM) cell lines as well as in 30 CMM tissue samples that were collected from the clinical service at our institution. We were able to confirm the expression of the MET protein in both melanoma cell lines, and we demonstrated MET activation by its ligand, HGF, through phosphorylation, in Western blot analysis. We were also able to demonstrate, by immunohistochemistry, the expression of MET in 63% of the tumor tissue samples analyzed, with the majority demonstrating a relatively low expression profile. We then evaluated the association of MET expression scores with histologic parameters, metastasis, and survival. While statistically significant associations were not found across these parameters, an inverse relationship between MET expression levels and time to lymph node versus distant metastasis was suggested in our cohort. These findings may require assessment in a larger group of specimens to further evaluate the role of MET expression in the homing of metastasis in lymph nodes versus that in distant organs.

BDNF is altered in a brain-region specific manner and rescues deficits in Spinocerebellar Ataxia Type 1.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset, dominantly inherited neurodegenerative disease caused by the expanded polyQ tract in the protein ATAXIN1 (ATXN1) and characterized by progressive motor and cognitive impairments. There are no disease-modifying treatments or cures for SCA1. Brain-derived neurotrophic factor (BDNF) plays important role in cerebellar physiology and has shown therapeutic potential for cerebellar pathology in the transgenic mouse model of SCA1, ATXN1[82Q] line that overexpress mutant ATXN1 under a cerebellar Purkinje-cell-specific promoter. Here we demonstrate decreased expression of brain derived neurotrophic factor (BDNF) in the cerebellum and medulla of patients with SCA1. Early stages of disease seem most amenable to therapy. Thus, we next quantified Bdnf expression in Atxn1154Q/2Q mice, a knock-in mouse model of SCA1, during the early symptomatic disease stage in four clinically relevant brain regions: cerebellum, medulla, hippocampus and motor cortex. We found that during the early stages of disease, Bdnf mRNA expression is reduced in the hippocampus and cerebellum, while it is increased in the cortex and brainstem. Importantly, we observed that pharmacological delivery of recombinant BDNF improved motor and cognitive performance, and mitigated pathology in the cerebellum and hippocampus of Atxn1154Q/2Q mice. Our findings demonstrate brain-region specific deficiency of BDNF in SCA1 and show that reversal of low BDNF levels offers the potential for meaningful treatment of motor and cognitive deficits in SCA1.

Blocking Site-Specific Cleavage of Human Tau Delays Progression of Disease-Related Phenotypes in Genetically Matched Tau-Transgenic Mice Modeling Frontotemporal Dementia.

Studies have recently demonstrated that a caspase-2-mediated cleavage of human tau (htau) at asparate-314 (D314) is responsible for cognitive deficits and neurodegeneration in mice modeling frontotemporal dementia (FTD). However, these animal studies may be confounded by flaws in their model systems, such as endogenous functional gene disruption and inequivalent transgene expression. To avoid these weaknesses, we examined the pathogenic role of this site-specific htau cleavage in FTD using genetically matched htau targeted-insertion mouse lines: rT2 and rT3. Both male and female mice were included in this study. rT2 mice contain a single copy of the FTD-linked htau proline-to-leucine mutation at amino acid 301 (htau P301L), inserted into a neutral site to avoid dysregulation of host gene expression. The similarly constructed rT3 mice harbor an additional D314-to-glutamate (D314E) mutation that blocks htau cleavage. We demonstrate that htau transgene expression occurs primarily in the forebrain at similar levels in rT2 and rT3 mice. Importantly, expression of the cleavage-resistant D314E mutant delays transgene-induced tau accumulation in the postsynaptic density, brain atrophy, hippocampal neurodegeneration, and spatial memory impairment, without altering age-related progression of pathologic tau conformation and phosphorylation. Our comprehensive investigation of age-dependent disease phenotypes associated with the htau P301L variant in precisely engineered FTD-modeling mice unveils a transiently protective effect of blocking htau cleavage at D314. Findings of this study advance our understanding of the contribution of this tau cleavage to the pathogenesis of FTD, and aid the development of effective dementia-targeting therapies.SIGNIFICANCE STATEMENT A site-specific and caspase-2-mediated cleavage of human tau plays a pathologic role in dementia. In this study, we investigate the contribution of this cleavage to the pathogenesis of frontotemporal dementia (FTD) using two genetically matched, tau-transgene targeted-insertion mouse lines that differ only by a cleavage-resistant mutation. The use of these mice avoids confounding effects associated with the random integration of tau transgenes to the mouse genome and allows us to comprehensively evaluate the impact of the tau cleavage on FTD phenotypes. Our data reveal that blocking this tau cleavage delays memory impairment and neurodegeneration of FTD-modeling mice. These findings improve our understanding of the pathogenic mechanisms underlying FTD and will facilitate the development of effective therapeutics.

UHRF2 regulates cell cycle, epigenetics and gene expression to control the timing of retinal progenitor and ganglion cell differentiation.

Ubiquitin-like, containing PHD and RING finger domains 2 (UHRF2) regulates cell cycle and binds 5-hydroxymethylcytosine (5hmC) to promote completion of DNA demethylation. Uhrf2-/- mice are without gross phenotypic defects; however, the cell cycle and epigenetic regulatory functions of Uhrf2 during retinal tissue development are unclear. Retinal progenitor cells (RPCs) produce all retinal neurons and Müller glia in a predictable sequence controlled by the complex interplay between extrinsic signaling, cell cycle, epigenetic changes and cell-specific transcription factor activation. In this study, we find that UHRF2 accumulates in RPCs, and its conditional deletion from mouse RPCs reduced 5hmC, altered gene expressions and disrupted retinal cell proliferation and differentiation. Retinal ganglion cells were overproduced in Uhrf2-deficient retinae at the expense of VSX2+ RPCs. Most other cell types were transiently delayed in differentiation. Expression of each member of the Tet3/Uhrf2/Tdg active demethylation pathway was reduced in Uhrf2-deficient retinae, consistent with locally reduced 5hmC in their gene bodies. This study highlights a novel role of UHRF2 in controlling the transition from RPCs to differentiated cell by regulating cell cycle, epigenetic and gene expression decisions.

Opposing transcriptional programs of KLF5 and AR emerge during therapy for advanced prostate cancer.

Endocrine therapies for prostate cancer inhibit the androgen receptor (AR) transcription factor. In most cases, AR activity resumes during therapy and drives progression to castration-resistant prostate cancer (CRPC). However, therapy can also promote lineage plasticity and select for AR-independent phenotypes that are uniformly lethal. Here, we demonstrate the stem cell transcription factor Krüppel-like factor 5 (KLF5) is low or absent in prostate cancers prior to endocrine therapy, but induced in a subset of CRPC, including CRPC displaying lineage plasticity. KLF5 and AR physically interact on chromatin and drive opposing transcriptional programs, with KLF5 promoting cellular migration, anchorage-independent growth, and basal epithelial cell phenotypes. We identify ERBB2 as a point of transcriptional convergence displaying activation by KLF5 and repression by AR. ERBB2 inhibitors preferentially block KLF5-driven oncogenic phenotypes. These findings implicate KLF5 as an oncogene that can be upregulated in CRPC to oppose AR activities and promote lineage plasticity.

Retinoblastoma tumor cell proliferation is negatively associated with an immune gene expression signature and increased immune cells.

This study focuses on gene expression differences between early retinal states that ultimately lead to normal development, late onset retinoblastoma, or rapid bilateral retinoblastoma tumors. The late-onset and early-onset retinoblastoma tumor cells are remarkably similar to normally proliferating retinal progenitor cells, but they fail to properly express differentiation markers associated with normal development. Further, early-onset retinoblastoma tumor cells express a robust immune gene expression signature followed by accumulation of dendritic, monocyte, macrophage, and T-lymphocyte cells in the retinoblastoma tumors. This characteristic was not shared by either normal retinae or late-onset retinoblastomas. Comparison of our data with other human and mouse retinoblastoma tumor gene expression significantly confirmed, that the immune signature is present in tumors from each species. Strikingly, we observed that the immune signature in both mouse and human tumors was most highly evident in those with the lowest proliferative capacity. We directly assessed this relationship in human retinoblastoma tumors by co-analyzing proliferation and immune cell recruitment by immunohistochemistry, uncovering a significant inverse relationship between increased immune-cell infiltration in tumors and reduced tumor cell proliferation. Directly inhibiting proliferation with a PI3K/mTOR inhibitor significantly increased the number of CD45+ immune cells in the retina. This work establishes an in vivo model for the rapid recruitment of immune cells to tumorigenic neural tissue.

Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions.

Sporadic angiosarcomas are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic angiosarcomas. In this study, we leveraged RNA-sequencing data from 13 human angiosarcomas and 76 spontaneous canine hemangiosarcomas to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, including TEX2-PECAM1 and ATP8A2-FLT1, were identified in seven of the 13 human tumors, with two tumors showing mutations of TP53. HRAS and NRAS mutations were found in angiosarcomas without fusions or TP53 mutations. We found 15 novel protein-coding fusion genes including MYO16-PTK2, GABRA3-FLT1, and AKT3-XPNPEP1 in 11 of the 76 canine hemangiosarcomas; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations in TP53, PIK3CA, PIK3R1, and NRAS. In particular, fusion genes and mutations of TP53 cooccurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human angiosarcomas and canine hemangiosarcomas identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data suggest that genome instability induced by TP53 mutations might create a predisposition for fusion events that may contribute to tumor progression by promoting selection and/or enhancing fitness through activation of convergent angiogenic pathways in this vascular malignancy. IMPLICATIONS: This study shows that, while drive events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphologic organization and biological behavior of these tumors in both species.

Single-cell RNA sequencing reveals that lung mesenchymal progenitor cells in IPF exhibit pathological features early in their differentiation trajectory.

In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

Metabolic reprogramming augments potency of human pSTAT3-inhibited iTregs to suppress alloreactivity.

Immunosuppressive donor Tregs can prevent graft-versus-host disease (GVHD) or solid-organ allograft rejection. We previously demonstrated that inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report that human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor antileukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing cutaneous lymphocyte-associated antigen, immunosuppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduced alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection and impaired infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provoked a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, cotreatment with coenzyme Q10 restored OxPhos in pSTAT3-inhibited iTregs and augmented their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.

A Rabbit Monoclonal Antibody against the Antiviral and Cancer Genomic DNA Mutating Enzyme APOBEC3B.

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.

Factors other than hTau overexpression that contribute to tauopathy-like phenotype in rTg4510 mice.

The tauopathy-like phenotype observed in the rTg4510 mouse line, in which human tauP301L expression specifically within the forebrain can be temporally controlled, has largely been attributed to high overexpression of mutant human tau in the forebrain region. Unexpectedly, we found that in a different mouse line with a targeted-insertion of the same transgene driven by the same tetracycline-TransActivator (tTA) allele, but with even higher overexpression of tauP301L than rTg4510, atrophy and tau histopathology are delayed, and a different behavioral profile is observed. This suggests that it is not overexpression of mutant human tau alone that contributes to the phenotype in rTg4510 mice. Furthermore we show that the tauopathy-like phenotype seen in rTg4510 requires a ~70-copy tau-transgene insertion in a 244 kb deletion in Fgf14, a ~7-copy tTA-transgene insertion in a 508 kb deletion that disrupts another five genes, in addition to high transgene overexpression. We propose that these additional effects need to be accounted for in any studies using rTg4510.

Registration of the extracellular matrix components constituting the fibroblastic focus in idiopathic pulmonary fibrosis.

The extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) drives fibrosis progression; however, the ECM composition of the fibroblastic focus (the hallmark lesion in IPF) and adjacent regions remains incompletely defined. Herein, we serially sectioned IPF lung specimens constructed into tissue microarrays and immunostained for ECM components reported to be deregulated in IPF. Immunostained sections were imaged, anatomically aligned, and 3D reconstructed. The myofibroblast core of the fibroblastic focus (defined by collagen I, α-smooth muscle actin, and procollagen I immunoreactivity) was associated with collagens III, IV, V, and VI; fibronectin; hyaluronan; and versican immunoreactivity. Hyaluronan immunoreactivity was also present at the fibroblastic focus perimeter and at sites where early lesions appear to be forming. Fibrinogen immunoreactivity was often observed at regions of damaged epithelium lining the airspace and the perimeter of the myofibroblast core but was absent from the myofibroblast core itself. The ECM components of the fibroblastic focus were distributed in a characteristic and reproducible manner in multiple patients. This information can inform the development of high-fidelity model systems to dissect mechanisms by which the IPF ECM drives fibrosis progression.

Amphiregulin in intestinal acute graft-versus-host disease: a possible diagnostic and prognostic aid.

Amphiregulin, a weak epidermal growth factor receptor agonist, is elevated, while epidermal growth factor, a strong epidermal growth factor receptor agonist, is low in the blood of patients with severe acute graft-versus-host disease. However, the tissue expression and function of these epidermal growth factor receptor ligands in acute graft-versus-host disease target organs is unknown. We compared by immunohistochemistry expression of amphiregulin and epidermal growth factor in archived, formalin-fixed, paraffin-embedded intestinal tissues of 48 patients with biopsy-proven gastrointestinal acute graft-versus-host disease to 3 groups: (1) 10 non-hematopoietic cell transplant normal controls, (2) 11 patients with newly diagnosed ulcerative colitis (ulcerative colitis), (3) 8 patients with a clinical diagnosis of acute graft-versus-host disease despite pathologically non-diagnostic biopsies, (4) and 10 cases of cytomegalovirus colitis. We used a semi-quantitative score of 0 (absent) through 3 (strong) to describe the intensity of immunohistochemical staining. We correlated serum and tissue amphiregulin and epidermal growth factor in patients with acute graft-versus-host disease. Gastrointestinal amphiregulin was significantly lower in acute graft-versus-host disease biopsies (median score 1), ulcerative colitis (median score 1.5), and cytomegalovirus colitis (median score 1) than in normal colon (median score 2, p = 0.004, p = 0.03, p = 0.009 respectively). Amphiregulin expression in was low in 74% of acute graft-versus-host disease cases with or without significant apoptosis. Patients with acute graft-versus-host disease exhibiting the pattern of high gastrointestinal amphiregulin but low serum amphiregulin (n = 14) had the best 1-year survival at 71%, but patients with high serum amphiregulin had poorer survival (<30%) regardless of gastrointestinal amphiregulin expression. Overall, our results lead to the hypothesis that amphiregulin is released into the circulation from damaged intestinal epithelium and stroma, although contributions from other cellular sources are likely. Low gastrointestinal amphiregulin expression by immunohistochemistry may be further studied for its utility in the pathologic acute graft-versus-host disease diagnosis without classic apoptotic changes.

The identification of a novel antibody for CD133 using human antibody phage display.

BACKGROUND: The transmembrane glycoprotein CD133 is believed to be a marker of adult prostate stem cells and cancer stem/initiating cells. Investigating the role of CD133 in the normal biology of the prostate and in cancer is complicated by the lack of a sensitive and accurate antibody for its detection. Here, we describe the characterization of a unique antibody identified using human antibody phage display that can recognize CD133 in both formalin-fixed tissues and cell lines. METHODS: A human single-chain variable fragment (scFv) antibody phage display library possessing a diversity of 8 × 109 was screened against fully glycosylated recombinant CD133. A counter screen was performed against deglycosylated CD133 to select for clones that preferentially recognized a glycosylation-independent epitope. The lead scFv was analyzed by flow cytometry and cloned into a rabbit immunoglobulin scaffold for immunohistochemistry (IHC). RESULTS: The antibody designated HA10 was found to bind a glycosylation-independent epitope on the peptide backbone of CD133 with high affinity. As a reagent for flow cytometry, HA10 detected CD133 more accurately than a commonly used commercially available antibody. IHC analysis with HA10 documented the staining of basal cells and luminal cells in healthy prostate sections. Weak staining of luminal cells was observed in adenocarcinoma sections at a very low frequency. Examination of a LuCaP patient-derived xenograft tissue microarray found that only three of the LuCaP models were positive for CD133. The three CD133pos LuCaP models all originated from non-AR driven metastatic prostate cancer with neuroendocrine differentiation. Subsequent interrogation of liver biopsies from a patient who failed second-generation anti-androgen therapy found high levels of CD133 staining. The original transurethral resection of the prostate from that patient was, however, absent of CD133. CONCLUSIONS: We have developed a novel antibody that was able to detect CD133 by both IHC and flow cytometry. Using HA10 as an IHC reagent, we found that CD133 is a marker for a very rare cell type in both healthy prostate and adenocarcinoma sections. Our preliminary investigation also suggests that there may be an association between CD133 and non-AR driven prostate cancer with neuroendocrine differentiation.

Dicer1 Deficiency in the Idiopathic Pulmonary Fibrosis Fibroblastic Focus Promotes Fibrosis by Suppressing MicroRNA Biogenesis.

RATIONALE: The lung extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) mediates progression of fibrosis by decreasing fibroblast expression of miR-29 (microRNA-29), a master negative regulator of ECM production. The molecular mechanism is undefined. IPF-ECM is stiffer than normal. Stiffness drives fibroblast ECM production in a YAP (yes-associated protein)-dependent manner, and YAP is a known regulator of miR-29. Therefore, we tested the hypothesis that negative regulation of miR-29 by IPF-ECM was mediated by mechanotransduction of stiffness. OBJECTIVES: To determine how IPF-ECM negatively regulates miR-29. METHODS: We decellularized lung ECM using detergents and prepared polyacrylamide hydrogels of defined stiffness by varying acrylamide concentrations. Mechanistic studies were guided by immunohistochemistry of IPF lung and used cell culture, RNA-binding protein assays, and xenograft models. MEASUREMENTS AND MAIN RESULTS: Contrary to our hypothesis, we excluded fibroblast mechanotransduction of ECM stiffness as the primary mechanism deregulating miR-29. Instead, systematic examination of miR-29 biogenesis revealed a microRNA processing defect that impeded processing of miR-29 into its mature bioactive forms. Immunohistochemical analysis of the microRNA processing machinery in IPF lung specimens revealed decreased Dicer1 expression in the procollagen-rich myofibroblastic core of fibroblastic foci compared with the focus perimeter and adjacent alveolar walls. Mechanistically, IPF-ECM increased association of the Dicer1 transcript with RNA binding protein AUF1 (AU-binding factor 1), and Dicer1 knockdown conferred primary human lung fibroblasts with cell-autonomous fibrogenicity in zebrafish and mouse lung xenograft models. CONCLUSIONS: Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.