Intravenous gentamicin therapy induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients: An open label clinical trial.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable widespread blistering skin disorder caused by mutations in the gene encoding for type VII collagen (C7), the major component of anchoring fibrils. OBJECTIVE: To evaluate the efficacy and safety of intravenous (IV) gentamicin-readthrough therapy in patients with RDEB harboring nonsense mutations. Primary outcomes were increased expression of C7 in patients' skin and assessments for safety (ototoxicity, nephrotoxicity, autoimmune response). Secondary outcomes included measuring wound healing in target wounds and assessment by a validated Epidermolysis Bullosa Disease and Activity Scarring Index (EBDASI) scoring system. METHODS: An open-label pilot trial assessing two different regimens of IV gentamicin between August 2018 and March 2020 with follow-up through 180 days post-treatment. Three RDEB patients with confirmed nonsense mutations in COL7A1 in either one or two alleles and decreased baseline expression of C7 at the dermal-epidermal junction (DEJ) of their skin participated in the study. Three patients received gentamicin at 7.5 mg/kg daily for 14 days and two of three patients further received 7.5 mg/kg IV gentamicin twice weekly for 12 weeks.Patients who had pre-existing auditory or renal impairment, were currently using ototoxic or nephrotoxic medications, or had allergies to aminoglycosides or sulfate compounds were excluded. RESULTS: After gentamicin treatment, skin biopsies from all three patients (ages ranging 18-28 years) exhibited increased C7 in their DEJ. With both regimens, the new C7 persisted at least six months post-treatment. At one and three-months post-treatment, 100% of the monitored wounds exhibited greater than 85% closure. Both IV gentamicin infusion regimens decreased EBDASI total activity scores. Of all patients assessed with the EBDASI, all patients exhibited decreased total activity scores three-month post-treatment. All three patients completed the study, and no adverse effects or anti-C7 antibodies were detected. CONCLUSIONS: IV gentamicin induced readthrough of nonsense mutations in RDEB patients and restored functional C7 in their skin, enhanced wound healing, and improved clinical parameters. IV gentamicin may be a safe, efficacious, low cost, and readily available therapy in this population of RDEB patients. TRIAL REGISTRATION: Clinicaltrials.gov Identifiers: NCT03392909.

Molecular and Clinical Outcomes After Intravenous Gentamicin Treatment for Patients With Junctional Epidermolysis Bullosa Caused by Nonsense Variants.

IMPORTANCE: Junctional epidermolysis bullosa (JEB) is an incurable blistering skin disorder with high infant mortality often caused by nonsense variants in the genes that encode laminin 332. OBJECTIVE: To evaluate the safety and outcomes following intravenous gentamicin readthrough therapy and subsequent laminin 332 expression in patients with JEB. DESIGN, SETTING, AND PARTICIPANTS: This open-label, pilot nonrandomized clinical trial assessed 1 course of low- or high-dose intravenous gentamicin, including follow-up at 30 and 90 days after treatment. Five pediatric patients with JEB (2 with intermediate JEB and 3 with severe JEB) and confirmed nonsense variants in LAMA3 or LAMB3 in 1 or 2 alleles and decreased expression of laminin 332 at the dermal-epidermal junction of their skin participated in the study, which was performed at a single institution in collaboration with physicians and home infusion services near the patients from April 1, 2019, to February 28, 2021, with follow-up until May 31, 2021. INTERVENTIONS: Three patients received gentamicin at 7.5 mg/kg daily for 14 days, and 2 patients received gentamicin at 10 mg/kg daily for 24 days. MAIN OUTCOMES AND MEASURES: Primary outcomes were change in expression of laminin 332 in patients' skin and assessments for safety (ototoxic effects, nephrotoxic effects, and autoimmune response). Secondary outcomes included wound healing in monitored wounds and Epidermolysis Bullosa Disease Activity and Scarring Index (EBDASI) score. RESULTS: After gentamicin treatment, all 5 patients (age range, 3 months to 10 years, 4 [80%] female) exhibited increased laminin 332 in the dermal-epidermal junction. By 1 month, 7 of 9 wounds in patients receiving low-dose intravenous gentamicin and all wounds in patients receiving high-dose intravenous gentamicin exhibited at least 50% wound closure. By 3 months, 8 of 9 wounds in patients receiving low-dose gentamicin and all wounds in patients receiving high-dose intravenous gentamicin exhibited greater than 85% closure. All 3 patients who were evaluated with EBDASI showed a decrease in total activity scores that met minimal clinically important differences 1 month after treatment. All 5 patients completed the study, and no ototoxic effects, nephrotoxic effects, or anti-laminin 332 antibodies were detected. CONCLUSIONS AND RELEVANCE: In this nonrandomized clinical trial, intravenous gentamicin therapy was associated with induced readthrough of nonsense variants in patients with JEB, restored functional laminin 332 in their skin, and wound closure during the 3-month study period. Although long-term safety and efficacy requires further evaluation, a single cycle of intravenous gentamicin may be a safe and readily available therapy in the short term for this population of patients with JEB. TRIAL REGISTRATION: ClinicalTrials.gov Identifiers: NCT03526159 and NCT04140786.

Characterization of mutant type VII collagens underlying the inversa subtype of recessive dystrophic epidermolysis bullosa.

BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen (C7) leading to skin fragility, bullae, and erosive wounds. RDEB-Inversa (RDEB-I), a subset of RDEB, is characterized by lesions localized to body areas with higher skin temperatures such as flexures and skin folds. OBJECTIVE: We aimed to determine if C7 derived from RDEB-I mutations had structural and functional aberrancies that were temperature sensitive and could be reversed by lowering the temperature. METHODS: In this study, we generated 12 substitution mutations associated with RDEB-I via site-directed mutagenesis and purified recombinant C7 protein. These C7 mutants were evaluated for structural parameters (trimer formation and protease sensitivity) and the ability to promote keratinocyte migration at 37 °C (the temperature of skin folds) and 30 °C (the maximum skin temperature of arms and legs). Fibroblasts derived from RDEB-I patients were evaluated for C7 secretion and cellular migration at both temperatures. RESULTS: C7s from RDEB-I mutations exhibited decreased thermal stability, increased sensitivity to protease digestion, diminished formation of collagen trimers, and reduced ability to promote keratinocyte migration compared with wild-type C7. In addition, fibroblasts derived from RDEB-I patients demonstrated intracellular accumulation of C7 and abnormal cell migration at 37 °C. All of these aberrancies were corrected by reducing the temperature to 30 °C. C7s generated from severe-RDEB mutations (non-Inversa) did not display temperature-dependent perturbations. CONCLUSION: These data demonstrate that RDEB-I mutations generate C7 aberrancies that are temperature dependent. This may explain why RDEB-I patients develop clinical lesions in areas where their skin is considerably warmer.

Over-expression of stromal periostin correlates with poor prognosis of cutaneous squamous cell carcinomas.

Periostin, an extracellular matrix macromolecule implicated in tumorigenesis, serves as a prognostic marker for many cancer types. However, there are no data on periostin expression in cutaneous squamous cell carcinoma (cSCC). This study examined periostin expression in patients with cSCC and explored its clincopathological relationship and prognosis. Using immunohistochemistry and ImageJ analysis, we compared periostin expression in 95 cSCCs across a spectrum of cSCC aggressiveness: cSCC in situ (SCCIS) (n = 25), low-risk cSCC (LR-cSCC) (n = 26), high-risk cSCC (HR-cSCC) (n = 38), and cSCC in recessive dystrophic epidermolysis bullosa patients (RDEB cSCC) (n = 6). Immunohistochemistry demonstrated periostin expression within the intra-tumoral stroma but not within tumor cells. Periostin levels significantly (P < 0.001) increased from SCCIS, LR-cSCC, HR-cSCC to RDEB SCC. The stroma of most of the cSCCs we evaluated contained cancer-associated fibroblasts with a myofibroblastic (α -SMA-positive) phenotype. Co-localization of periostin with α-SMA, evidence of fibroblast periostin expression, and absence of keratinocyte or tumor cell periostin expression suggest that, in cSCC, periostin is a product of the peritumoral microenvironment and not the tumor cells themselves. Our data indicate that fibroblast periostin expression is highly correlated with the aggressiveness of cSCC, and may thereby provide a molecular marker that will be useful for subtyping and diagnosing cSCCs according to their biological nature.

Gentamicin Induces Laminin 332 and Improves Wound Healing in Junctional Epidermolysis Bullosa Patients with Nonsense Mutations.

Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3, LAMB3, or LAMC2 genes. Most of these mutations are nonsense mutations that create premature termination codons that lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients' skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least 3 months and were associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients' blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.

Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin ß3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin ß3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin ß3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin ß3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6ß4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

Gentamicin induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable disease caused by mutations in the gene encoding type VII collagen, the major component of anchoring fibrils (AF). We previously demonstrated that gentamicin produced functional type VII collagen in RDEB cells harboring nonsense mutations. Herein, we determined whether topical or intradermal gentamicin administration induces type VII collagen and AFs in RDEB patients. METHODS: A double-blind, placebo-controlled pilot trial assessed safety and efficacy of topical and intradermal gentamicin in 5 RDEB patients with nonsense mutations. The topical arm tested 0.1% gentamicin ointment or placebo application 3 times daily at 2 open erosion sites for 2 weeks. The intradermal arm tested daily intradermal injection of gentamicin solution (8 mg) or placebo into 2 intact skin sites for 2 days in 4 of 5 patients. Primary outcomes were induction of type VII collagen and AFs at the test sites and safety assessment. A secondary outcome assessed wound closure of topically treated erosions. RESULTS: Both topical and intradermal gentamicin administration induced type VII collagen and AFs at the dermal-epidermal junction of treatment sites. Newly created type VII collagen varied from 20% to 165% of that expressed in normal human skin and persisted for 3 months. Topical gentamicin corrected dermal-epidermal separation, improved wound closure, and reduced blister formation. There were no untoward side effects from gentamicin treatments. Type VII collagen induction did not generate anti-type VII collagen autoantibodies in patients' blood or skin. CONCLUSION: Topical and intradermal gentamicin suppresses nonsense mutations and induces type VII collagen and AFs in RDEB patients. Gentamicin therapy may provide a readily available treatment for RDEB patients with nonsense mutations. TRIAL REGISTRATION: ClinicalTrials.gov NCT02698735. FUNDING: Epidermolysis Bullosa Research Partnership, Epidermolysis Bullosa Medical Research Foundation, NIH, and VA Merit Award.

Intravenously Administered Recombinant Human Type VII Collagen Derived from Chinese Hamster Ovary Cells Reverses the Disease Phenotype in Recessive Dystrophic Epidermolysis Bullosa Mice.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder characterized by skin fragility, blistering, and multiple skin wounds with no currently approved or consistently effective treatment. It is due to mutations in the gene encoding type VII collagen (C7). Using recombinant human C7 (rhC7) purified from human dermal fibroblasts (FB-rhC7), we showed previously that intravenously injected rhC7 distributed to engrafted RDEB skin, incorporated into its dermal-epidermal junction (DEJ), and reversed the RDEB disease phenotype. Human dermal fibroblasts, however, are not used for commercial production of therapeutic proteins. Therefore, we generated rhC7 from Chinese hamster ovary (CHO) cells. The CHO-derived recombinant type VII collagen (CHO-rhC7), similar to FB-rhC7, was secreted as a correctly folded, disulfide-bonded, helical trimer resistant to protease degradation. CHO-rhC7 bound to fibronectin and promoted human keratinocyte migration in vitro. A single dose of CHO-rhC7, administered intravenously into new-born C7-null RDEB mice, incorporated into the DEJ of multiple skin sites, tongue and esophagus, restored anchoring fibrils, improved dermal-epidermal adherence, and increased the animals' life span. Furthermore, no circulating or tissue-bound anti-C7 antibodies were observed in the mice. These data demonstrate the efficacy of CHO-rhC7 in a preclinical murine model of RDEB.

Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.

Patients with recessive dystrophic epidermolysis bullosa (RDEB) have severe, incurable skin fragility, blistering, and multiple skin wounds due to mutations in the gene encoding type VII collagen (C7), the major component of anchoring fibrils mediating epidermal-dermal adherence. Nearly 10-25% of RDEB patients carry nonsense mutations leading to premature stop codons (PTCs) that result in truncated C7. In this study, we evaluated the feasibility of using aminoglycosides to suppress PTCs and induce C7 expression in two RDEB keratinocyte cell lines (Q251X/Q251X and R578X/R906) and two primary RDEB fibroblasts (R578X/R578X and R163X/R1683X). Incubation of these cells with aminoglycosides (geneticin, gentamicin, and paromomycin) resulted in the synthesis and secretion of a full-length C7 in a dose-dependent and sustained manner. Importantly, aminoglycoside-induced C7 reversed the abnormal RDEB cell phenotype and incorporated into the dermal-epidermal junction of skin equivalents. We further demonstrated the general utility of aminoglycoside-mediated readthrough in 293 cells transiently transfected with expression vectors encoding 22 different RDEB nonsense mutations. This is the first study demonstrating that aminoglycosides can induce PTC readthrough and restore functional C7 in RDEB caused by nonsense mutations. Therefore, aminoglycosides may have therapeutic potential for RDEB patients and other inherited skin diseases caused by nonsense mutations.

Krox20/EGR2 deficiency accelerates cell growth and differentiation in the monocytic lineage and decreases bone mass.

Krox20/EGR2, one of the 4 early growth response genes, is a highly conserved transcription factor implicated in hindbrain development, peripheral nerve myelination, tumor suppression, and monocyte/macrophage cell fate determination. Here, we established a novel role for Krox20 in postnatal skeletal metabolism. Microcomputed tomographic analysis of 4- and 8-week-old mice revealed a low bone mass phenotype (LBM) in both the distal femur and the vertebra of Krox20(+/-) mice. This was attributable to accelerated bone resorption as demonstrated in vivo by increased osteoclast number and serum C-terminal telopeptides, a marker for collagen degradation. Krox20 haploinsufficiency did not reduce bone formation in vivo, nor did it compromise osteoblast differentiation in vitro. In contrast, growth and differentiation were significantly stimulated in preosteoclast cultures derived from Krox20(+/-) splenocytes, suggesting that the LBM is attributable to Krox20 haploinsufficiency in the monocytic lineage. Furthermore, Krox20 silencing in preosteoclasts increased cFms expression and response to macrophage colony-stimulating factor, leading to a cell-autonomous stimulation of cell-cycle progression. Our data indicate that the antimitogenic role of Krox20 in preosteoclasts is the predominant mechanism underlying the LBM phenotype of Krox20-deficient mice. Stimulation of Krox20 expression in preosteoclasts may present a viable therapeutic strategy for high-turnover osteoporosis.

Lef1 haploinsufficient mice display a low turnover and low bone mass phenotype in a gender- and age-specific manner.

We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1(+/-) females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3beta, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR) (tfm mutants). The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1(+/-) female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.

Inhibition of AR-mediated transcription by binding of Oct1 to a motif enriched in AR-occupied regions.

BACKGROUND: The androgen receptor (AR) plays roles in prostate development and cancer (PCa). In response to androgens, the AR binds to androgen-response elements (AREs) to modulate gene transcription. The responses of such genes are dependent on the cellular milieu and on sequences around the AREs, which attract other transcription factors. Previously, bioinformatic analysis of 62 AR-occupied regions (ARORs) in PCa cells revealed enrichment for both AREs and a TTGGCAAATA-like motif. We undertook the present study to investigate the significance of the TTGGCAAATA-like motif. METHODS: Prostate cancer cell lines, LNCaP and C4-2B, were analyzed by transient transfections of wild-type and mutant reporter constructs, electro-mobility shift assays (EMSAs), and RT-qPCR analysis of endogenous genes. RESULTS: In two of six tested ARORs, point mutations in the TTGGCAAATA-like motif resulted in inhibition of DHT-mediated enhancer activity. EMSA revealed that Oct1 bound the motif, and that the mutations that abolished DHT responsiveness in the transfection assays augmented Oct1 binding. These results suggest a role for Oct1 as a context-dependent negative coregulator of AR. Consistent with this, siRNA knockdown of Oct1 increased the DHT-mediated enhancer activity of transfected reporters as well as an endogenous AR target gene, transglutaminase 2. CONCLUSIONS: Oct1 negatively regulates DHT-mediated enhancer activity in a subset of ARORs. The enrichment of ARORs for the Oct-binding, TTGGCAAATA-like motif may reflect a mechanism that utilizes Oct1 to keep AR activity in check at some ARORs, while augmenting AR activity in other ARORs. Therefore, Oct1 may have regulatory functions in prostate development and cancer progression.

Genomic androgen receptor-occupied regions with different functions, defined by histone acetylation, coregulators and transcriptional capacity.

BACKGROUND: The androgen receptor (AR) is a steroid-activated transcription factor that binds at specific DNA locations and plays a key role in the etiology of prostate cancer. While numerous studies have identified a clear connection between AR binding and expression of target genes for a limited number of loci, high-throughput elucidation of these sites allows for a deeper understanding of the complexities of this process. METHODOLOGY/PRINCIPAL FINDINGS: We have mapped 189 AR occupied regions (ARORs) and 1,388 histone H3 acetylation (AcH3) loci to a 3% continuous stretch of human genomic DNA using chromatin immunoprecipitation (ChIP) microarray analysis. Of 62 highly reproducible ARORs, 32 (52%) were also marked by AcH3. While the number of ARORs detected in prostate cancer cells exceeded the number of nearby DHT-responsive genes, the AcH3 mark defined a subclass of ARORs much more highly associated with such genes -- 12% of the genes flanking AcH3+ARORs were DHT-responsive, compared to only 1% of genes flanking AcH3-ARORs. Most ARORs contained enhancer activities as detected in luciferase reporter assays. Analysis of the AROR sequences, followed by site-directed ChIP, identified binding sites for AR transcriptional coregulators FoxA1, CEBPbeta, NFI and GATA2, which had diverse effects on endogenous AR target gene expression levels in siRNA knockout experiments. CONCLUSIONS/SIGNIFICANCE: We suggest that only some ARORs function under the given physiological conditions, utilizing diverse mechanisms. This diversity points to differential regulation of gene expression by the same transcription factor related to the chromatin structure.

Opposing effects of glucocorticoids and Wnt signaling on Krox20 and mineral deposition in osteoblast cultures.

Krox20 is expressed in osteoblasts and chondrocytes, and is required for trabecular bone formation during embryogenesis. Here we show by RT-qPCR and Western blot analysis that Krox20 is up-regulated during late stages of osteoblast differentiation in culture. Glucocorticoids (GCs) rapidly inhibit the expression of Krox20 as well its co-activator, HCF-1, resulting in inhibition of the Osteocalcin Krox20-binding Enhancer (OKE). GCs also inhibit expression of EGR1, EGR3, and EGR4. OKE activity, which is dependent on the presence of Runx2, was independent of the osteocalcin promoter Runx2 binding site. In contrast to GCs, activation of the Wnt, but not the BMP or the PTH signaling pathways, stimulated Krox20 expression as well as activity of the OKE. GC-mediated suppression of Krox20 expression was compromised, albeit not completely, in the presence of DKK1, suggesting that the inhibition occurs in both Wnt-dependent and Wnt-independent manners. Furthermore, Wnt3A partially rescued Krox20 expression in GC-arrested osteoblast cultures and this was accompanied by rescue of mineralization. These findings are consistent with a role for Krox20 in osteoblast function and suggest that this transcription factor may contribute to the opposing effects of GCs and Wnt signaling on bone formation.

Androgen receptor-mediated repression of novel target genes.

BACKGROUND: The androgen receptor (AR) plays a pivotal role in prostate cancer (PCa) initiation and progression. To date, studies have focused disproportionately on androgen-stimulated genes such as prostate-specific antigen (PSA), while repressed genes have gained little attention, even though they too may be involved in regulating cell growth, differentiation, and apoptosis. METHODS: ChIP Display was used to identify putative AR target genes in the ablation-resistant human PCa cell line, C4-2B. Quantitative real-time reverse transcription-PCR analysis was used to measure gene expression in cells subjected to dihydrotestosterone (DHT) timecourse and dose-response, as well as AR knock-down and bicalutamide-treatments. RESULTS: We report on three genes, KIAA1217, CHRM1, and WBSCR28, which were newly identified in a screen for AR-occupied regions in C4-2B PCa cells, and which were repressed by treatment with DHT. AR knock-down resulted in increased KIAA1217, CHRM1, and WBSCR28 mRNA, indicating that, like PSA stimulation, AR represses these three genes even in the absence of added ligand. DHT decreased KIAA1217 and CHRM1 pre-mRNA levels, suggesting AR-mediated transcriptional inhibition. Cycloheximide attenuated DHT-mediated repression of CHRM1, suggesting the requirement of new protein synthesis. Furthermore, bicalutamide treatment did not mimic, but rather antagonized DHT-mediated KIAA1217 repression. Unlike the handful of androgen-repressed genes studied thus far, AR occupancy at KIAA1217, CHRM1, and WBSCR28 was mapped outside their respective 5'-promoter regions. CONCLUSIONS: Many more genes likely share AR-mediated gene repression through distal regulatory elements. Further study of such targets and their transcriptional regulation may help explain the receptor's tumorigenicity in PCa.

Identification of novel androgen receptor target genes in prostate cancer.

BACKGROUND: The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression. RESULTS: Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells--D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1)--most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation. CONCLUSION: AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.