Increased expression of desmin and vimentin reduces bladder smooth muscle contractility via JNK2.

Bladder dysfunction is associated with the overexpression of the intermediate filament (IF) proteins desmin and vimentin in obstructed bladder smooth muscle (BSM). However, the mechanisms by which these proteins contribute to BSM dysfunction are not known. Previous studies have shown that desmin and vimentin directly participate in signal transduction. In this study, we hypothesized that BSM dysfunction associated with overexpression of desmin or vimentin is mediated via c-Jun N-terminal kinase (JNK). We employed a model of murine BSM tissue in which increased expression of desmin or vimentin was induced by adenoviral transduction to examine the sufficiency of increased IF protein expression to reduce BSM contraction. Murine BSM strips overexpressing desmin or vimentin generated less force in response to KCl and carbachol relative to the levels in control murine BSM strips, an effect associated with increased JNK2 phosphorylation and reduced myosin light chain (MLC20 ) phosphorylation. Furthermore, desmin and vimentin overexpressions did not alter BSM contractility and MLC20 phosphorylation in strips isolated from JNK2 knockout mice. Pharmacological JNK2 inhibition produced results qualitatively similar to those caused by JNK2 knockout. These findings suggest that inhibition of JNK2 may improve diminished BSM contractility associated with obstructive bladder disease.

Dark Side of Cancer Therapy: Cancer Treatment-Induced Cardiopulmonary Inflammation, Fibrosis, and Immune Modulation.

Advancements in cancer therapy increased the cancer free survival rates and reduced the malignant related deaths. Therapeutic options for patients with thoracic cancers include surgical intervention and the application of chemotherapy with ionizing radiation. Despite these advances, cancer therapy-related cardiopulmonary dysfunction (CTRCPD) is one of the most undesirable side effects of cancer therapy and leads to limitations to cancer treatment. Chemoradiation therapy or immunotherapy promote acute and chronic cardiopulmonary damage by inducing reactive oxygen species, DNA damage, inflammation, fibrosis, deregulation of cellular immunity, cardiopulmonary failure, and non-malignant related deaths among cancer-free patients who received cancer therapy. CTRCPD is a complex entity with multiple factors involved in this pathogenesis. Although the mechanisms of cancer therapy-induced toxicities are multifactorial, damage to the cardiac and pulmonary tissue as well as subsequent fibrosis and organ failure seem to be the underlying events. The available biomarkers and treatment options are not sufficient and efficient to detect cancer therapy-induced early asymptomatic cell fate cardiopulmonary toxicity. Therefore, application of cutting-edge multi-omics technology, such us whole-exome sequencing, DNA methylation, whole-genome sequencing, metabolomics, protein mass spectrometry and single cell transcriptomics, and 10 X spatial genomics, are warranted to identify early and late toxicity, inflammation-induced carcinogenesis response biomarkers, and cancer relapse response biomarkers. In this review, we summarize the current state of knowledge on cancer therapy-induced cardiopulmonary complications and our current understanding of the pathological and molecular consequences of cancer therapy-induced cardiopulmonary fibrosis, inflammation, immune suppression, and tumor recurrence, and possible treatment options for cancer therapy-induced cardiopulmonary toxicity.

NF-κB and GATA-Binding Factor 6 Repress Transcription of Caveolins in Bladder Smooth Muscle Hypertrophy.

Caveolins (CAVs) are structural proteins of caveolae that function as signaling platforms to regulate smooth muscle contraction. Loss of CAV protein expression is associated with impaired contraction in obstruction-induced bladder smooth muscle (BSM) hypertrophy. In this study, microarray analysis of bladder RNA revealed down-regulation of CAV1, CAV2, and CAV3 gene transcription in BSM from models of obstructive bladder disease in mice and humans. We identified and characterized regulatory regions responsible for CAV1, CAV2, and CAV3 gene expression in mice with obstruction-induced BSM hypertrophy, and in men with benign prostatic hyperplasia. DNA affinity chromatography and chromatin immunoprecipitation assays revealed a greater increase in binding of GATA-binding factor 6 (GATA-6) and NF-κB to their cognate binding motifs on CAV1, CAV2, and CAV3 promoters in obstructed BSM relative to that observed in control BSM. Knockout of NF-κB subunits, shRNA-mediated knockdown of GATA-6, or pharmacologic inhibition of GATA-6 and NF-κB in BSM increased CAV1, CAV2, and CAV3 transcription and promoter activity. Conversely, overexpression of GATA-6 decreased CAV2 and CAV3 transcription and promoter activity. Collectively, these data provide new insight into the mechanisms by which CAV gene expression is repressed in hypertrophied BSM in obstructive bladder disease.

Improvement in Therapeutic Efficacy and Reduction in Cellular Toxicity: Introduction of a Novel Anti-PSMA-Conjugated Hybrid Antiandrogen Nanoparticle.

Second generation antiandrogens have improved overall survival for men with metastatic castrate resistant prostate cancer; however, the antiandrogens result in suppression of androgen receptor (AR) activity in all tissues resulting in dose limiting toxicity. We sought to overcome this limitation through encapsulation in a prostate specific membrane antigen (PSMA)-conjugated nanoparticle. We designed and characterized a novel nanoparticle containing an antiandrogen, enzalutamide. Selectivity and enhanced efficacy was achieved through coating the particle with PSMA. The PSMA-conjugated nanoparticle was internalized selectively in AR expressing prostate cancer cells. It did not elicit an inflammatory effect. The efficacy of enzalutamide was not compromised through insertion into the nanoparticle; in fact, lower systemic drug concentrations of enzalutamide resulted in comparable clinical activity. Normal muscle cells were not impacted by the PSMA-conjugated containing antiandrogen. This approach represents a novel strategy to increase the specificity and effectiveness of antiandrogen treatment for men with castrate resistant prostate cancer. The ability to deliver higher drug concentrations in prostate cancer cells may translate into improved clinical end points including overall survival.

Aging-associated changes in microRNA expression profile of internal anal sphincter smooth muscle: Role of microRNA-133a.

A comprehensive genomic and proteomic, computational, and physiological approach was employed to examine the (previously unexplored) role of microRNAs (miRNAs) as regulators of internal anal sphincter (IAS) smooth muscle contractile phenotype and basal tone. miRNA profiling, genome-wide expression, validation, and network analyses were employed to assess changes in mRNA and miRNA expression in IAS smooth muscles from young vs. aging rats. Multiple miRNAs, including rno-miR-1, rno-miR-340-5p, rno-miR-185, rno-miR-199a-3p, rno-miR-200c, rno-miR-200b, rno-miR-31, rno-miR-133a, and rno-miR-206, were found to be upregulated in aging IAS. qPCR confirmed the upregulated expression of these miRNAs and downregulation of multiple, predicted targets (Eln, Col3a1, Col1a1, Zeb2, Myocd, Srf, Smad1, Smad2, Rhoa/Rock2, Fn1, Tagln v2, Klf4, and Acta2) involved in regulation of smooth muscle contractility. Subsequent studies demonstrated an aging-associated increase in the expression of miR-133a, corresponding decreases in RhoA, ROCK2, MYOCD, SRF, and SM22α protein expression, RhoA-signaling, and a decrease in basal and agonist [U-46619 (thromboxane A2 analog)]-induced increase in the IAS tone. Moreover, in vitro transfection of miR-133a caused a dose-dependent increase of IAS tone in strips, which was reversed by anti-miR-133a. Last, in vivo perianal injection of anti-miR-133a reversed the loss of IAS tone associated with age. This work establishes the important regulatory effect of miRNA-133a on basal and agonist-stimulated IAS tone. Moreover, reversal of age-associated loss of tone via anti-miR delivery strongly implicates miR dysregulation as a causal factor in the aging-associated decrease in IAS tone and suggests that miR-133a is a feasible therapeutic target in aging-associated rectoanal incontinence.

Bimodal protein targeting through activation of cryptic mitochondrial targeting signals by an inducible cytosolic endoprotease.

Bimodal targeting of the endoplasmic reticular protein, cytochrome P4501A1 (CYP1A1), to mitochondria involves activation of a cryptic mitochondrial targeting signal through endoprotease processing of the protein. Here, we characterized the endoprotease that regulates mitochondrial targeting of CYP1A1. The endoprotease, which was induced by beta-naphthoflavone, was a dimer of 90 kDa and 40 kDa subunits, each containing Ser protease domains. The purified protease processed CYP1A1 in a sequence-specific manner, leading to its mitochondrial import. The glucocorticoid receptor, retinoid X receptor, and p53 underwent similar processing-coupled mitochondrial transport. The inducible 90 kDa subunit was a limiting factor in many cells and some tissues and, thus, regulates the mitochondrial levels of these proteins. A number of other mitochondria-associated proteins with noncanonical targeting signals may also be substrates of this endoprotease. Our results describe a new mechanism of mitochondrial protein import that requires an inducible cytoplasmic endoprotease for activation of cryptic mitochondrial targeting signals.

Mechanical stretch upregulates proteins involved in Ca2+ sensitization in urinary bladder smooth muscle hypertrophy.

Partial bladder outlet obstruction (pBOO)-induced remodeling of bladder detrusor smooth muscle (DSM) is associated with the modulation of cell signals regulating contraction. We analyzed the DSM from obstructed murine urinary bladders for the temporal regulation of RhoA GTPase and Rho-activated kinase (ROCK), which are linked to Ca(2+) sensitization. In addition, the effects of equibiaxial cell stretch, a condition thought to be associated with pBOO-induced bladder wall smooth muscle hypertrophy and voiding frequency, on the expression of RhoA, ROCK, and C-kinase-activated protein phosphatase I inhibitor (CPI-17) were investigated. DSM from 1-, 3-, 7-, and 14-day obstructed male mice bladders and benign prostatic hyperplasia (BPH)-induced obstructed human bladders revealed overexpression of RhoA and ROCK-ß at the mRNA and protein levels compared with control. Primary human bladder myocytes seeded onto type I collagen-coated elastic silicone membranes were subjected to cyclic equibiaxial stretch, mimicking the cellular mechanical stretch in the bladder in vivo, and analyzed for the expression of RhoA, ROCK-ß, and CPI-17. Stretch caused a significant increase of RhoA, ROCKß, and CPI-17 expression. The stretch-induced increase in CPI-17 expression occurs at the transcriptional level and is associated with CPI-17 promoter binding by GATA-6 and NF-κB, the transcription factors responsible for CPI-17 gene transcription. Cell stretch caused by bladder overdistension in pBOO is the likely mechanism for initiating overexpression of the signaling proteins regulating DSM tone.

Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle.

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

Innate Immune System in the Context of Radiation Therapy for Cancer.

Radiation therapy (RT) remains an integral component of modern oncology care, with most cancer patients receiving radiation as a part of their treatment plan. The main goal of ionizing RT is to control the local tumor burden by inducing DNA damage and apoptosis within the tumor cells. The advancement in RT, including intensity-modulated RT (IMRT), stereotactic body RT (SBRT), image-guided RT, and proton therapy, have increased the efficacy of RT, equipping clinicians with techniques to ensure precise and safe administration of radiation doses to tumor cells. In this review, we present the technological advancement in various types of RT methods and highlight their clinical utility and associated limitations. This review provides insights into how RT modulates innate immune signaling and the key players involved in modulating innate immune responses, which have not been well documented earlier. Apoptosis of cancer cells following RT triggers immune systems that contribute to the eradication of tumors through innate and adoptive immunity. The innate immune system consists of various cell types, including macrophages, dendritic cells, and natural killer cells, which serve as key mediators of innate immunity in response to RT. This review will concentrate on the significance of the innate myeloid and lymphoid lineages in anti-tumorigenic processes triggered by RT. Furthermore, we will explore essential strategies to enhance RT efficacy. This review can serve as a platform for researchers to comprehend the clinical application and limitations of various RT methods and provides insights into how RT modulates innate immune signaling.

Transcriptional repression of Caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.

Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits down-regulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Silencing of GATA6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in bladder smooth muscle cells. Together, these data suggest that GATA-6 acts as a transcriptional repressor of CAV1 gene expression in PBOO-induced BSM hypertrophy in men and mice. GATA-6-induced transcriptional repression represents a new regulatory mechanism of CAV1 gene expression in pathologic BSM, and may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men.

Bone Health Management in the Continuum of Prostate Cancer Disease.

Prostate cancer (PCa) is the second-leading cause of cancer-related deaths in men. PCa cells require androgen receptor (AR) signaling for their growth and survival. Androgen deprivation therapy (ADT) is the preferred treatment for patients with locally advanced and metastatic PCa disease. Despite their initial response to androgen blockade, most patients eventually will develop metastatic castration-resistant prostate cancer (mCRPC). Bone metastases are common in men with mCRPC, occurring in 30% of patients within 2 years of castration resistance and in >90% of patients over the course of the disease. Patients with mCRPC-induced bone metastasis develop lesions throughout their skeleton; the 5-year survival rate for these patients is 47%. Bone-metastasis-induced early changes in the bone that proceed the osteoblastic response in the bone matrix are monitored and detected via modern magnetic resonance and PET/CT imaging technologies. Various treatment options, such as targeting osteolytic metastasis with bisphosphonates, prednisone, dexamethasone, denosumab, immunotherapy, external beam radiation therapy, radiopharmaceuticals, surgery, and pain medications are employed to treat prostate-cancer-induced bone metastasis and manage bone health. However, these diagnostics and treatment options are not very accurate nor efficient enough to treat bone metastases and manage bone health. In this review, we present the pathogenesis of PCa-induced bone metastasis, its deleterious impacts on vital organs, the impact of metastatic PCa on bone health, treatment interventions for bone metastasis and management of bone- and skeletal-related events, and possible current and future therapeutic options for bone management in the continuum of prostate cancer disease.

Alterations in caveolin expression and ultrastructure after bladder smooth muscle hypertrophy.

PURPOSE: Partial bladder outlet obstruction in male rabbits causes detrusor smooth muscle hypertrophy and voiding dysfunction similar to that observed in men with benign prostate hyperplasia. Using this model, we analyzed the protein expression and ultrastructure of caveolae and the intermediate size filament in detrusor smooth muscle following partial bladder outlet obstruction induced hypertrophy. MATERIALS AND METHODS: Detrusor smooth muscle sections from bladder body were processed for immunofluorescence and electron microscopy. Western analysis was performed to determine the expression of caveolin isoform-1, 2 and 3, and intermediate size filament proteins. RESULTS: Detrusor smooth muscle cells from both normal and hypertrophied bladders contain orderly arrays of thick and thin myofilaments, interspersed with dense bodies. In addition, there was an increase in intermediate size filaments in the hypertrophic detrusor smooth muscle cells. The dense plaques in the inner membrane of hypertrophied detrusor smooth muscle were longer than those of the control. Detrusor smooth muscle from hypertrophied bladder revealed a decreased number of caveolae and a lack of their orderly distribution at the plasma membrane. Western blotting showed decreased expression of caveolin-1, 2 and 3 in hypertrophied detrusor smooth muscle. CONCLUSIONS: Caveolae serve as platforms for proteins and receptors that have a role in signal transduction. The decreased number of caveolae and caveolin protein expression in hypertrophied detrusor smooth muscle might contribute to alterations in signal transduction pathways that regulate the downstream effects of agonist induced contraction, including calcium sensitization, observed in obstructed bladder. In addition, the increased number of intermediate size filaments in the hypertrophied detrusor smooth muscle is likely to alter the cytoskeletal structure and affect the cellular transmission of passive and/or active force.

Therapeutic Challenge with a CDK 4/6 Inhibitor Induces an RB-Dependent SMAC-Mediated Apoptotic Response in Non-Small Cell Lung Cancer.

Purpose: The retinoblastoma tumor suppressor (RB), a key regulator of cell-cycle progression and proliferation, is functionally suppressed in up to 50% of non-small cell lung cancer (NSCLC). RB function is exquisitely controlled by a series of proteins, including the CyclinD-CDK4/6 complex. In this study, we interrogated the capacity of a CDK4/6 inhibitor, palbociclib, to activate RB function.Experimental Design and Results: We employed multiple isogenic RB-proficient and -deficient NSCLC lines to interrogate the cytostatic and cytotoxic capacity of CDK 4/6 inhibition in vitro and in vivo We demonstrate that while short-term exposure to palbociclib induces cellular senescence, prolonged exposure results in inhibition of tumor growth. Mechanistically, CDK 4/6 inhibition induces a proapoptotic transcriptional program through suppression of IAPs FOXM1 and Survivin, while simultaneously augmenting expression of SMAC and caspase-3 in an RB-dependent manner.Conclusions: This study uncovers a novel function of RB activation to induce cellular apoptosis through therapeutic administration of a palbociclib and provides a rationale for the clinical evaluation of CDK 4/6 inhibitors in the treatment of patients with NSCLC. Clin Cancer Res; 24(6); 1402-14. ©2018 AACR.

RB Loss Promotes Prostate Cancer Metastasis.

RB loss occurs commonly in neoplasia but its contributions to advanced cancer have not been assessed directly. Here we show that RB loss in multiple murine models of cancer produces a prometastatic phenotype. Gene expression analyses showed that regulation of the cell motility receptor RHAMM by the RB/E2F pathway was critical for epithelial-mesenchymal transition, motility, and invasion by cancer cells. Genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes induced by RB loss in prostate cancer. Mechanistic studies in this setting established that RHAMM stabilized F-actin polymerization by controlling ROCK signaling. Collectively, our findings show how RB loss drives metastatic capacity and highlight RHAMM as a candidate therapeutic target for treating advanced prostate cancer. Cancer Res; 77(4); 982-95. ©2016 AACR.

A synthetic peptide with estrogen-like activity derived from a phage-display peptide library.

We describe a novel approach to develop peptides with estrogen like activity using a monoclonal antibody specific to estradiol (mAb E2-15) for the affinity selection of phage displayed peptides from a combinatorial peptide library. Based on the sequences of the selected phage, we synthesized a 15-mer linear peptide LPALDPTKRWFFETK which was derivatized to a 23 mer cyclic peptide CAELPALDPTKRWFFETKPPPPC. Both peptides displayed estrogen-like activity according to the following criteria:(i) in inhibiting the binding of [3H]estradiol to mAb E2-15 and to estrogen receptor (ER)alpha; (ii) in inducing transcriptional activity in MCF7 human breast cancer cells transfected with an estrogen receptor element luciferase construct and (iii) in causing an increase in creatine kinase specific activity in rat tissues in vivo. This approach can be employed to design peptide mimetic for other hormones as well.

The retinoblastoma tumor suppressor modulates DNA repair and radioresponsiveness.

PURPOSE: Perturbations in the retinoblastoma pathway are over-represented in advanced prostate cancer; retinoblastoma loss promotes bypass of first-line hormone therapy. Conversely, preliminary studies suggested that retinoblastoma-deficient tumors may become sensitized to a subset of DNA-damaging agents. Here, the molecular and in vivo consequence of retinoblastoma status was analyzed in models of clinical relevance. EXPERIMENTAL DESIGN: Experimental work was performed with multiple isogenic prostate cancer cell lines (hormone sensitive: LNCaP and LAPC4 cells and hormone resistant C42, 22Rv1 cells; stable knockdown of retinoblastoma using shRNA). Multiple mechanisms were interrogated including cell cycle, apoptosis, and DNA damage repair. Transcriptome analysis was performed, validated, and mechanisms discerned. Cell survival was measured using clonogenic cell survival assay and in vivo analysis was performed in nude mice with human derived tumor xenografts. RESULTS: Loss of retinoblastoma enhanced the radioresponsiveness of both hormone-sensitive and castrate-resistant prostate cancer. Hypersensitivity to ionizing radiation was not mediated by cell cycle or p53. Retinoblastoma loss led to alteration in DNA damage repair and activation of the NF-κB pathway and subsequent cellular apoptosis through PLK3. In vivo xenografts of retinoblastoma-deficient tumors exhibited diminished tumor mass, lower PSA kinetics, and decreased tumor growth after treatment with ionizing radiation (P < 0.05). CONCLUSIONS: Loss of retinoblastoma confers increased radiosensitivity in prostate cancer. This hypersensitization was mediated by alterations in apoptotic signaling. Combined, these not only provide insight into the molecular consequence of retinoblastoma loss, but also credential retinoblastoma status as a putative biomarker for predicting response to radiotherapy.

Inherent sex-dependent regulation of human hepatic CYP3A5.

BACKGROUND AND PURPOSE: Expression of hepatic cytochromes P450 (CYP) in all species examined, including humans, is generally sexually dimorphic. We examined the sex-dependent expression of CYP3A5 and the hormone-regulated molecular mechanism(s) responsible for any dimorphism. EXPERIMENTAL APPROACH: CYP3A5 levels as well as nuclear translocation and promoter binding of transcription factors regulating CYP3A5 expression were measured in primary hepatocyte cultures derived from men and women exposed to physiological-like levels of growth hormone alone, dexamethasone alone and the combined regimen. KEY RESULTS: We observed a dramatic inherent CYP3A5 sexual dimorphism (women > men) with all treatments as a result of a ~2-fold greater level of hormone-induced activation and nuclear accumulation of hepatocyte nuclear factor-4α (HNF-4α), pregnane X receptor (PXR) and retinoic X receptorα (RXRα) in female hepatocytes. Furthermore, PXR : RXRα exhibited significantly higher DNA binding levels to its specific binding motif on the CYP3A5 promoter in female hepatocytes, inferring a possible explanation for the elevated expression of the isoform in women. Results from experiments using HepG2 cells treated with siRNA-induced knockdown of HNF-4α and/or transfected with luciferase reporter constructs containing the CYP3A5 promoter were in agreement with the basic mechanism observed in primary hepatocytes of both sexes. CONCLUSIONS AND IMPLICATIONS: Female-predominant expression of human CYP3A5 is due to an inherent, sex-dependent suboptimal activation of the transcription networks responsible for hormone-induced expression of the isoform in men. Accordingly, in conjunction with previous studies of other human CYPs, men and women are intrinsically unlikely to handle many drugs in the same way; thus, sex should be a requisite component factored into the design of personalized drug therapies.

Regulation of miR106b cluster through the RB pathway: mechanism and functional targets.

The RB pathway plays a critical role in proliferation control that is commonly subverted in tumor development. However, restoration of RB pathway function can be elicited in many tumor cells by the inhibition of CDK4/6 activity that leads to dephosphorylation of RB and subsequent repression of E2F-mediated transcription. In this context, active RB/E2F complexes inhibit the expression of a critical program of coding genes that promote cell cycle progression. However, the non-coding RNA target genes downstream from RB that could be relevant for tumor biology remain obscure. Here, miRNA gene expression profiling identified the miR106b cluster as being efficiently repressed with CDK4/6 inhibition in an E2F and RB-dependent manner. Importantly, the miR106B-cluster is intragenic of MCM7, and through a series of functional studies, the basis of MCM7 regulation and concordant expression of the miRNA species within the 106b cluster was determined. Importantly, RB-mediated repression of the 106b cluster enhances the transcript levels of p21Cip1 and PTEN. These data provide a mechanistic basis for cross-talk between the RB pathway and p21 and PTEN through the regulation of the MCM7/miR106b locus.

GATA-6 and NF-κB activate CPI-17 gene transcription and regulate Ca2+ sensitization of smooth muscle contraction.

Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca(2+) sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH.