SERPINE1: Role in Cholangiocarcinoma Progression and a Therapeutic Target in the Desmoplastic Microenvironment.

A heterogenous population of inflammatory elements, other immune and nonimmune cells and cancer-associated fibroblasts (CAFs) are evident in solid malignancies where they coexist with the growing tumor mass. In highly desmoplastic malignancies, CAFs are the prominent mesenchymal cell type in the tumor microenvironment (TME), where their presence and abundance signal a poor prognosis. CAFs play a major role in the progression of various cancers by remodeling the supporting stroma into a dense, fibrotic matrix while secreting factors that promote the maintenance of cancer stem-like characteristics, tumor cell survival, aggressive growth and metastasis and reduced sensitivity to chemotherapeutics. Tumors with high stromal fibrotic signatures are more likely to be associated with drug resistance and eventual relapse. Identifying the molecular underpinnings for such multidirectional crosstalk among the various normal and neoplastic cell types in the TME may provide new targets and novel opportunities for therapeutic intervention. This review highlights recent concepts regarding the complexity of CAF biology in cholangiocarcinoma, a highly desmoplastic cancer. The discussion focuses on CAF heterogeneity, functionality in drug resistance, contributions to a progressively fibrotic tumor stroma, the involved signaling pathways and the participating genes.

Downstream Targets of VHL/HIF-α Signaling in Renal Clear Cell Carcinoma Progression: Mechanisms and Therapeutic Relevance.

The clear cell variant of renal cell carcinoma (ccRCC) is the most common renal epithelial malignancy and responsible for most of the deaths from kidney cancer. Patients carrying inactivating mutations in the Von Hippel-Lindau (VHL) gene have an increased proclivity to develop several types of tumors including ccRCC. Normally, the Hypoxia Inducible Factor alpha (HIF-α) subunits of the HIF heterodimeric transcription factor complex are regulated by oxygen-dependent prolyl-hydroxylation, VHL-mediated ubiquitination and proteasomal degradation. Loss of pVHL function results in elevated levels of HIF-α due to increased stability, leading to RCC progression. While HIF-1α acts as a tumor suppressor, HIF-2α promotes oncogenic potential by driving tumor progression and metastasis through activation of hypoxia-sensitive signaling pathways and overexpression of HIF-2α target genes. One strategy to suppress ccRCC aggressiveness is directed at inhibition of HIF-2α and the associated molecular pathways leading to cell proliferation, angiogenesis, and metastasis. Indeed, clinical and pre-clinical data demonstrated the effectiveness of HIF-2α targeted therapy in attenuating ccRCC progression. This review focuses on the signaling pathways and the involved genes (cyclin D, c-Myc, VEGF-a, EGFR, TGF-α, GLUT-1) that confer oncogenic potential downstream of the VHL-HIF-2α signaling axis in ccRCC. Discussed as well are current treatment options (including receptor tyrosine kinase inhibitors such as sunitinib), the medical challenges (high prevalence of metastasis at the time of diagnosis, refractory nature of advanced disease to current treatment options), scientific challenges and future directions.

Emerging role of tumor suppressor p53 in acute and chronic kidney diseases.

p53 is a major regulator of cell cycle arrest, apoptosis, and senescence. While involvement of p53 in tumorigenesis is well established, recent studies implicate p53 in the initiation and progression of several renal diseases, which is the focus of this review. Ischemic-, aristolochic acid (AA) -, diabetic-, HIV-associated-, obstructive- and podocyte-induced nephropathies are accompanied by activation and/or elevated expression of p53. Studies utilizing chemical or renal-specific inhibition of p53 in mice confirm the pathogenic role of this transcription factor in acute kidney injury and chronic kidney disease. TGF-ß1, NOX, ATM/ATR kinases, Cyclin G, HIPK, MDM2 and certain micro-RNAs are important determinants of renal p53 function in response to trauma. AA, cisplatin or TGF-ß1-mediated ROS generation via NOXs promotes p53 phosphorylation and subsequent tubular dysfunction. p53-SMAD3 transcriptional cooperation downstream of TGF-ß1 orchestrates induction of fibrotic factors, extracellular matrix accumulation and pathogenic renal cell communication. TGF-ß1-induced micro-RNAs (such as mir-192) could facilitate p53 activation, leading to renal hypertrophy and matrix expansion in response to diabetic insults while AA-mediated mir-192 induction regulates p53 dependent epithelial G2/M arrest. The widespread involvement of p53 in tubular maladaptive repair, interstitial fibrosis, and podocyte injury indicate that p53 clinical targeting may hold promise as a novel therapeutic strategy for halting progression of certain acute and chronic renal diseases, which affect hundreds of million people worldwide.

Cancer-Associated Fibroblasts: Mechanisms of Tumor Progression and Novel Therapeutic Targets.

Cancer-associated fibroblasts (CAFs) are a heterogenous population of stromal cells found in solid malignancies that coexist with the growing tumor mass and other immune/nonimmune cellular elements. In certain neoplasms (e.g., desmoplastic tumors), CAFs are the prominent mesenchymal cell type in the tumor microenvironment, where their presence and abundance signal a poor prognosis in multiple cancers. CAFs play a major role in the progression of various malignancies by remodeling the supporting stromal matrix into a dense, fibrotic structure while secreting factors that lead to the acquisition of cancer stem-like characteristics and promoting tumor cell survival, reduced sensitivity to chemotherapeutics, aggressive growth and metastasis. Tumors with high stromal fibrotic signatures are more likely to be associated with drug resistance and eventual relapse. Clarifying the molecular basis for such multidirectional crosstalk among the various normal and neoplastic cell types present in the tumor microenvironment may yield novel targets and new opportunities for therapeutic intervention. This review highlights the most recent concepts regarding the complexity of CAF biology including CAF heterogeneity, functionality in drug resistance, contribution to a progressively fibrotic tumor stroma, the involved signaling pathways and the participating genes.

Heat Shock Protein 27, a Novel Downstream Target of Collagen Type XI alpha 1, Synergizes with Fatty Acid Oxidation to Confer Cisplatin Resistance in Ovarian Cancer Cells.

Collagen type XI alpha 1 (COL11A1) is a novel biomarker associated with cisplatin resistance in ovarian cancer. We have previously reported that COL11A1 activates Src-Akt signaling through the collagen receptors discoidin domain receptor 2 (DDR2) and integrin α1ß1 to confer cisplatin resistance to ovarian cancer cells. To identify the potential signaling molecules downstream of COL11A1 signaling, we performed protein kinase arrays and identified heat shock protein 27 (HSP27) as a potential mediator of COL11A1-induced cisplatin resistance. Through receptor knockdown and inhibitor experiments, we demonstrated that COL11A1 significantly upregulates HSP27 phosphorylation and expression via DDR2/integrin α1ß1 and Src/Akt signaling in ovarian cancer cells. Furthermore, genetic knockdown and pharmacological inhibition of HSP27, via ivermectin treatment, significantly sensitizes ovarian cancer cells cultured on COL11A1 to cisplatin treatment. HSP27 knockdown or inhibition also decreases NFκB activity as well as the expression of inhibitors of apoptosis proteins (IAPs), which are known downstream effector molecules of COL11A1 that promote cisplatin resistance. Interestingly, HSP27 knockdown or inhibition stimulates ovarian cancer cells to upregulate fatty acid oxidation (FAO) for survival and cisplatin resistance, and dual inhibition of HSP27 and FAO synergistically kills ovarian cancer cells that are cultured on COL11A1. Collectively, this study identifies HSP27 as a novel and druggable COL11A1 downstream effector molecule that may be targeted to overcome cisplatin resistance in recurrent ovarian cancer, which often overexpress COL11A1.

PAI-1 induction during kidney injury promotes fibrotic epithelial dysfunction via deregulation of klotho, p53, and TGF-ß1-receptor signaling.

Renal fibrosis leads to chronic kidney disease, which affects over 15% of the U.S. population. PAI-1 is highly upregulated in the tubulointerstitial compartment in several common nephropathies and PAI-1 global ablation affords protection from fibrogenesis in mice. The precise contribution of renal tubular PAI-1 induction to disease progression, however, is unknown and surprisingly, appears to be independent of uPA inhibition. Human renal epithelial (HK-2) cells engineered to stably overexpress PAI-1 underwent dedifferentiation (E-cadherin loss, gain of vimentin), G2/M growth arrest (increased p-Histone3, p21), and robust induction of fibronectin, collagen-1, and CCN2. These cells are also susceptible to apoptosis (elevated cleaved caspase-3, annexin-V positivity) compared to vector controls, demonstrating a previously unknown role for PAI-1 in tubular dysfunction. Persistent PAI-1 expression results in a loss of klotho expression, p53 upregulation, and increases in TGF-ßRI/II levels and SMAD3 phosphorylation. Ectopic restoration of klotho in PAI-1-transductants attenuated fibrogenesis and reversed the proliferative defects, implicating PAI-1 in klotho loss in renal disease. Genetic suppression of p53 reversed the PA1-1-driven maladaptive repair, moreover, confirming a pathogenic role for p53 upregulation in this context and uncovering a novel role for PAI-1 in promoting renal p53 signaling. TGF-ßRI inhibition also attenuated PAI-1-initiated epithelial dysfunction, independent of TGF-ß1 ligand synthesis. Thus, PAI-1 promotes tubular dysfunction via klotho reduction, p53 upregulation, and activation of the TGF-ßRI-SMAD3 axis. Since klotho is an upstream regulator of both PAI-1-mediated p53 induction and SMAD3 signaling, targeting tubular PAI-1 expression may provide a novel, multi-level approach to the therapy of CKD.

Negative regulators of TGF-ß1 signaling in renal fibrosis; pathological mechanisms and novel therapeutic opportunities.

Elevated expression of the multifunctional cytokine transforming growth factor ß1 (TGF-ß1) is causatively linked to kidney fibrosis progression initiated by diabetic, hypertensive, obstructive, ischemic and toxin-induced injury. Therapeutically relevant approaches to directly target the TGF-ß1 pathway (e.g., neutralizing antibodies against TGF-ß1), however, remain elusive in humans. TGF-ß1 signaling is subjected to extensive negative control at the level of TGF-ß1 receptor, SMAD2/3 activation, complex assembly and promoter engagement due to its critical role in tissue homeostasis and numerous pathologies. Progressive kidney injury is accompanied by the deregulation (loss or gain of expression) of several negative regulators of the TGF-ß1 signaling cascade by mechanisms involving protein and mRNA stability or epigenetic silencing, further amplifying TGF-ß1/SMAD3 signaling and fibrosis. Expression of bone morphogenetic proteins 6 and 7 (BMP6/7), SMAD7, Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene (SnoN), phosphate tensin homolog on chromosome 10 (PTEN), protein phosphatase magnesium/manganese dependent 1A (PPM1A) and Klotho are dramatically decreased in various nephropathies in animals and humans albeit with different kinetics while the expression of Smurf1/2 E3 ligases are increased. Such deregulations frequently initiate maladaptive renal repair including renal epithelial cell dedifferentiation and growth arrest, fibrotic factor (connective tissue growth factor (CTGF/CCN2), plasminogen activator inhibitor type-1 (PAI-1), TGF-ß1) synthesis/secretion, fibroproliferative responses and inflammation. This review addresses how loss of these negative regulators of TGF-ß1 pathway exacerbates renal lesion formation and discusses the therapeutic value in restoring the expression of these molecules in ameliorating fibrosis, thus, presenting novel approaches to suppress TGF-ß1 hyperactivation during chronic kidney disease (CKD) progression.

Protein phosphatase Mg2+ /Mn2+ dependent-1A and PTEN deregulation in renal fibrosis: Novel mechanisms and co-dependency of expression.

PPM1A and PTEN emerged as novel suppressors of chronic kidney disease (CKD). Since loss of PPM1A and PTEN in the tubulointerstitium promotes fibrogenesis, defining molecular events underlying PPM1A/PTEN deregulation is necessary to develop expression rescue as novel therapeutic strategies. Here we identify TGF-ß1 as a principle repressor of PPM1A, as conditional renal tubular-specific induction of TGF-ß1 in mice dramatically downregulates kidney PPM1A expression. TGF-ß1 similarly attenuates PPM1A and PTEN expression in human renal epithelial cells and fibroblasts, via a protein degradation mechanism by promoting their ubiquitination. A proteasome inhibitor MG132 rescues PPM1A and PTEN expression, even in the presence of TGF-ß1, along with decreased fibrogenesis. Restoration of PPM1A or PTEN similarly limits SMAD3 phosphorylation and the activation of TGF-ß1-induced fibrotic genes. Concurrent loss of PPM1A and PTEN levels in aristolochic acid nephropathy further suggests crosstalk between these repressors. PPM1A silencing in renal fibroblasts, moreover, results in PTEN loss, while PTEN stable depletion decreases PPM1A expression with acquisition of a fibroproliferative phenotype in each case. Transient PPM1A expression, conversely, elevates cellular PTEN levels while lentiviral PTEN introduction increases PPM1A expression. PPM1A and PTEN, therefore, co-regulate each other's relative abundance, identifying a previously unknown pathological link between TGF-ß1 repressors, contributing to CKD.

The TGF-ß1/p53/PAI-1 Signaling Axis in Vascular Senescence: Role of Caveolin-1.

Stress-induced premature cellular senescence is a significant factor in the onset of age-dependent disease in the cardiovascular system. Plasminogen activator inhibitor-1 (PAI-1), a major TGF-ß1/p53 target gene and negative regulator of the plasmin-based pericellular proteolytic cascade, is elevated in arterial plaques, vessel fibrosis, arteriosclerosis, and thrombosis, correlating with increased tissue TGF-ß1 levels. Additionally, PAI-1 is necessary and sufficient for the induction of p53-dependent replicative senescence. The mechanism of PAI-1 transcription in senescent cells appears to be dependent on caveolin-1 signaling. Src kinases are upstream effectors of both FAK and caveolin-1 activation as FAKY577,Y861 and caveolin-1Y14 phosphorylation are not detected in TGF-ß1-stimulated src family kinase (pp60c-src, Yes, Fyn) triple-deficient (SYF-/-/-) cells. However, restoration of pp60c-src expression in SYF-null cells rescued both caveolin-1Y14 phosphorylation and PAI-1 induction in response to TGF-ß1. Furthermore, TGF-ß1-initiated Src phosphorylation of caveolin-1Y14 is critical in Rho-ROCK-mediated suppression of the SMAD phosphatase PPM1A maintaining and, accordingly, SMAD2/3-dependent transcription of the PAI-1 gene. Importantly, TGF-ß1 failed to induce PAI-1 expression in caveolin-1-null cells, correlating with reductions in both Rho-GTP loading and SMAD2/3 phosphorylation. These findings implicate caveolin-1 in expression controls on specific TGF-ß1/p53 responsive growth arrest genes. Indeed, up-regulation of caveolin-1 appears to stall cells in G0/G1 via activation of the p53/p21 cell cycle arrest pathway and restoration of caveolin-1 in caveolin-1-deficient cells rescues TGF-ß1 inducibility of the PAI-1 gene. Although the mechanism is unclear, caveolin-1 inhibits p53/MDM2 complex formation resulting in p53 stabilization, induction of p53-target cell cycle arrest genes (including PAI-1), and entrance into premature senescence while stimulating the ATM→p53→p21 pathway. Identification of molecular events underlying senescence-associated PAI-1 expression in response to TGF-ß1/src kinase/p53 signaling may provide novel targets for the therapy of cardiovascular disease.

TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

Chronic kidney disease affects >15% of the U.S. population and >850 million individuals worldwide. Fibrosis is the common outcome of many chronic renal disorders and, although the etiology varies (i.e., diabetes, hypertension, ischemia, acute injury, and urologic obstructive disorders), persistently elevated renal TGF-ß1 levels result in the relentless progression of fibrotic disease. TGF-ß1 orchestrates the multifaceted program of renal fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery and redifferentiation, and subsequent tubulointerstitial fibrosis, eventually leading to chronic renal disease. Recent findings implicate p53 as a cofactor in the TGF-ß1-induced signaling pathway and a transcriptional coregulator of several TGF-ß1 profibrotic response genes by complexing with receptor-activated SMADs, which are homologous to the small worms (SMA) and Drosophilia mothers against decapentaplegic (MAD) gene families. The cooperative p53-TGF-ß1 genomic cluster includes genes involved in cell growth control and extracellular matrix remodeling [e.g., plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor, clade E, member 1), connective tissue growth factor, and collagen I]. Although the molecular basis for this codependency is unclear, many TGF-ß1-responsive genes possess p53 binding motifs. p53 up-regulation and increased p53 phosphorylation; moreover, they are evident in nephrotoxin- and ischemia/reperfusion-induced injury, diabetic nephropathy, ureteral obstructive disease, and kidney allograft rejection. Pharmacologic and genetic approaches that target p53 attenuate expression of the involved genes and mitigate the fibrotic response, confirming a key role for p53 in renal disorders. This review focuses on mechanisms whereby p53 functions as a transcriptional regulator within the TGF-ß1 cluster with an emphasis on the potent fibrosis-promoting PAI-1 gene.-Higgins, C. E., Tang, J., Mian, B. M., Higgins, S. P., Gifford, C. C., Conti, D. J., Meldrum, K. K., Samarakoon, R., Higgins, P. J. TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

Rac-GTPase promotes fibrotic TGF-ß1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways.

Rac-GTPases are major regulators of cytoskeletal remodeling and their deregulation contributes to numerous pathologies. Whether or how Rac promotes tubulointerstitial fibrosis and chronic kidney disease (CKD) is currently unknown. We showed that the major profibrotic cytokine, TGF-ß1 promoted rapid Rac1-GTP loading in human kidney 2 (HK-2) human renal epithelial cells. A Rac-specific chemical inhibitor, EHT 1864, blocked TGF-ß1-induced fibrotic reprogramming in kidney epithelial cells and fibroblasts. Stable Rac1 depletion in HK-2 cells, moreover, eliminated TGF-ß1-mediated non-SMAD pathway activation [e.g., Src, epidermal growth factor receptor (EGFR), p53] and subsequent plasminogen activator inhibitor-1 (PAI-1), connective tissue growth factor, fibronectin, and p21 induction. Rac1 and p22phox knockdown abrogated free radical generation by TGF-ß1 in HK-2 cells, consistent with the role of Rac1 in NAPD(H). TGF-ß1-induced renal epithelial cytostasis was also completely bypassed by Rac1, p22phox, p47phox, and PAI-1 silencing. Rac1b isoform expression was robustly induced in the fibrotic kidneys of mice and humans. Intraperitoneal administration of EHT 1864 in mice dramatically attenuated ureteral unilateral obstruction-driven EGFR, p53, Rac1b, yes-associated protein/transcriptional coactivator with PDZ-binding motif activation/expression, dedifferentiation, cell cycle arrest, and renal fibrogenesis evident in vehicle-treated obstructed kidneys. Thus, the Rac1-directed redox response is critical for TGF-ß1-driven epithelial dysfunction orchestrated, in part, via PAI-1 up-regulation. Rac pathway inhibition suppressed renal oxidative stress and maladaptive repair, identifying Rac as a novel therapeutic target against progressive CKD.-Patel, S., Tang, J., Overstreet, J. M., Anorga, S., Lian, F., Arnouk, A., Goldschmeding, R., Higgins, P. J., Samarakoon, R. Rac-GTPase promotes fibrotic TGF-ß1 signaling and chronic kidney disease via EGFR, p53, and Hippo/YAP/TAZ pathways.

Deregulation of Negative Controls on TGF-ß1 Signaling in Tumor Progression.

The multi-functional cytokine transforming growth factor-ß1 (TGF-ß1) has growth inhibitory and anti-inflammatory roles during homeostasis and the early stages of cancer. Aberrant TGF-ß activation in the late-stages of tumorigenesis, however, promotes development of aggressive growth characteristics and metastatic spread. Given the critical importance of this growth factor in fibrotic and neoplastic disorders, the TGF-ß1 network is subject to extensive, multi-level negative controls that impact receptor function, mothers against decapentaplegic homolog 2/3 (SMAD2/3) activation, intracellular signal bifurcation into canonical and non-canonical pathways and target gene promotor engagement. Such negative regulators include phosphatase and tensin homologue (PTEN), protein phosphatase magnesium 1A (PPM1A), Klotho, bone morphogenic protein 7 (BMP7), SMAD7, Sloan-Kettering Institute proto-oncogene/ Ski related novel gene (Ski/SnoN), and bone morphogenetic protein and activin membrane-bound Inhibitor (BAMBI). The progression of certain cancers is accompanied by loss of expression, overexpression, mislocalization, mutation or deletion of several endogenous repressors of the TGF-ß1 cascade, further modulating signal duration/intensity and phenotypic reprogramming. This review addresses how their aberrant regulation contributes to cellular plasticity, tumor progression/metastasis and reversal of cell cycle arrest and discusses the unexplored therapeutic value of restoring the expression and/or function of these factors as a novel approach to cancer treatment.

Deregulation of Hippo-TAZ pathway during renal injury confers a fibrotic maladaptive phenotype.

Although yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), nuclear transducers of the Hippo pathway, are mostly silent in adult organs, aberrant activation of YAP/TAZ promotes tumorigenesis and abnormal tissue repair. The extent of involvement of TAZ in chronic kidney disease (CKD) is unknown. In our study, increased TAZ nuclear accumulation and expression in the tubulointerstitium was readily evident in 3 models of renal injury including obstructive, aristolochic acid (AA), and diabetic nephropathy, correlating with fibrosis progression. Stable TAZ overexpression in human kidney (HK)-2 epithelial cells promoted connective tissue growth factor (CTGF), fibronectin, vimentin, and p21 expression, epithelial dedifferentiation, and growth inhibition, in part, via Sma mothers against decapentaplegic homologue (SMAD)-3-dependent CTGF induction. CTGF secretion by TAZ-overexpressing epithelium also triggered proliferative defects in nonengineered HK-2 cells confirming a nonautonomous role of TAZ ( via a paracrine mechanism) in orchestrating kidney epithelial cell-cell communication. Renal tubular-specific induction of TGF-ß1 in mice and TGF-ß1 stimulation of HK-2 cells resulted in TAZ protein up-regulation. TAZ stable silencing in HK-2 cells abrogated TGF-ß1-induced expression of target genes without affecting SMAD3 phosphorylation, which is also crucial for fibrotic reprogramming. Thus, TAZ was activated in fibrosis through TGF-ß1-dependent mechanisms and sustained TAZ signaling promotes epithelial maladaptive repair. TAZ is also a novel non-SMAD downstream effector of renal TGF-ß1 signaling, establishing TAZ as a new antifibrosis target for treatment of CKD.-Anorga, S., Overstreet, J. M., Falke, L. L., Tang, J., Goldschmeding, R. G., Higgins, P. J., Samarakoon, R. Deregulation of Hippo-TAZ pathway during renal injury confers a fibrotic maladaptive phenotype.

The Cytoskeletal Network Regulates Expression of the Profibrotic Genes PAI-1 and CTGF in Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are subject to changing hemodynamic stimuli that alter cytoskeletal dynamics, cellular architecture, and structure-associated signal transduction. Tensional stress, force application, and structural perturbations are sensed by VSMCs and impact the physiological as well as pathophysiological responses of the vasculature. Microtubule-targeting drugs provide useful tools to analyze cytoskeletal-associated signaling pathways and their linkages to pathological outcomes. Architecture-based controls on a subset of profibrotic genes commonly expressed in vascular disease are highlighted by their frequent induction in mechanically manipulated cells and with associated changes in cytoskeletal dynamics. VSMCs respond to biomechanical cues by activating several kinase cascades, leading to gene reprogramming. It is apparent that a significant fraction of the vast repertoire of signaling intermediates, moreover, are sequestered on the cytoskeletal framework in an "inactive state." Reorganization within these networks due to fluctuating mechanical forces could release these effectors from their cytoskeletal anchors, thus alleviating the "repressive state" resulting in downstream signaling. Indeed, recent findings indicate that microtubule disruption in VSMCs rapidly stimulates pp60c-src kinase activation and epidermal growth factor receptor (EGFR) transphosphorylation at Y845, a src kinase target residue. EGFR genetic deficiency, pharmacological inhibition of EGFR signaling, or adenoviral delivery of the kinase-deficient EGFRK721A construct effectively blocked colchicine-stimulated expression of two prominent vascular profibrotic genes, plasminogen activator inhibitor type-1 (PAI-1; SERPINE1) and connective tissue growth factor (CTGF; CCN2). Signaling intermediates involved in microtubule collapse-initiated PAI-1/CTGF induction in VSMCs include the MEK/ERK, Rho/ROCK, and SMAD2/3 pathways. This review highlights commonalities and differences in signaling events that facilitate expression of vascular disease-relevant genes initiated as a consequence of loss of microtubular integrity.

TGF-ß1/p53 signaling in renal fibrogenesis.

Fibrotic disorders of the renal, pulmonary, cardiac, and hepatic systems are associated with significant morbidity and mortality. Effective therapies to prevent or curtail the advancement to organ failure, however, remain a major clinical challenge. Chronic kidney disease, in particular, constitutes an increasing medical burden affecting >15% of the US population. Regardless of etiology (diabetes, hypertension, ischemia, acute injury, urologic obstruction), persistently elevated TGF-ß1 levels are causatively linked to the activation of profibrotic signaling networks and disease progression. TGF-ß1 is the principal driver of renal fibrogenesis, a dynamic pathophysiologic process that involves tubular cell injury/apoptosis, infiltration of inflammatory cells, interstitial fibroblast activation and excess extracellular matrix synthesis/deposition leading to impaired kidney function and, eventually, to chronic and end-stage disease. TGF-ß1 activates the ALK5 type I receptor (which phosphorylates SMAD2/3) as well as non-canonical (e.g., src kinase, EGFR, JAK/STAT, p53) pathways that collectively drive the fibrotic genomic program. Such multiplexed signal integration has pathophysiological consequences. Indeed, TGF-ß1 stimulates the activation and assembly of p53-SMAD3 complexes required for transcription of the renal fibrotic genes plasminogen activator inhibitor-1, connective tissue growth factor and TGF-ß1. Tubular-specific ablation of p53 in mice or pifithrin-α-mediated inactivation of p53 prevents epithelial G2/M arrest, reduces the secretion of fibrotic effectors and attenuates the transition from acute to chronic renal injury, further supporting the involvement of p53 in disease progression. This review focuses on the pathophysiology of TGF-ß1-initiated renal fibrogenesis and the role of p53 as a regulator of profibrotic gene expression.

Integration of Canonical and Noncanonical Pathways in TLR4 Signaling: Complex Regulation of the Wound Repair Program.

Significance: Chronic inflammation and maladaptive repair contribute to the development of fibrosis that negatively impacts quality of life and organ function. The toll-like receptor (TLR) system is a critical node in the tissue response to both exogenous (pathogen-associated) and endogenous (damage-associated) molecular pattern factors (PAMPs and DAMPs, respectively). The development of novel TLR ligand-, pathway-, and/or target gene-specific therapeutics may have clinical utility in the management of the exuberant inflammatory/fibrotic tissue response to injury without compromising the host defense to pathogens. Recent Advances: DAMP ligands, released upon wounding, and microbial-derived PAMPs interact with several TLRs, and their various coreceptor partners, engaging downstream pathways that include Src family kinases, the epidermal growth factor receptor, integrins and the tumor suppressor phosphatase and tensin homolog (PTEN). Toll-like receptor 4 (TLR4) activation enhances cellular responses to the potent profibrotic cytokine transforming growth factor-ß1 (TGF-ß1) by attenuating the expression of receptors that inhibit TGF-ß1 signaling. Critical Issues: Common as well as unique pathways may be activated by PAMP and DAMP ligands that bind to the repertoire of TLRs on various cell types. Dissecting mechanisms underlying ligand-dependent engagement of this complex, highly interactive, network will provide for adaptation of new and focused therapies directed to the regulation of pathologically significant profibrotic genes. Inherent in this diversity are therapeutic opportunities to modulate the pathophysiologic consequences of persistent TLR signaling. The recently identified involvement of receptor and nonreceptor kinase pathways in TLR signaling may present novel opportunities for pharmacologic intervention. Future Directions: Clarifying the identity and function of DAMP-activated TLR complexes or ligand-binding partners, as well as their engaged downstream effectors and target genes, are key factors in the eventual design of pathway-specific treatment modalities. Such approaches may be tailored to address the spectrum of TLR-initiated pathologies (including localized and persistent inflammation, maladaptive repair/fibrosis) and, perhaps, even titrated to achieve patient-unique beneficial clinical outcomes.

Insights into cellular and molecular basis for urinary tract infection in autosomal-dominant polycystic kidney disease.

Urinary tract infection (UTI) is a broad term referring to an infection of the kidneys, ureters, bladder, and/or urethra. Because of its prevalence, frequent recurrence, and rising resistance to antibiotics, UTI has become a challenge in clinical practice. Autosomal-dominant polycystic kidney disease (ADPKD) is the most common monogenic disorder of the kidney and is characterized by the growth of fluid-filled cysts in both kidneys. Progressive cystic enlargement, inflammation, and interstitial fibrosis result in nephron loss with subsequent decline in kidney function. ADPKD patients frequently develop UTI; however, the cellular and molecular mechanisms responsible for the high UTI incidence in ADPKD patients remain virtually unaddressed. Emerging evidence suggests that α-intercalated cells (α-ICs) of the collecting ducts function in the innate immune defense against UTI. α-ICs inhibit bacterial growth by acidifying urine and secreting neutrophil gelatinase-associated lipocalin (NGAL) that chelates siderophore-containing iron. It is necessary to determine, therefore, if ADPKD patients with recurrent UTI have a reduced number and/or impaired function of α-ICs. Identification of the underlying cellular and molecular mechanisms may lead to the development of novel strategies to reduce UTI in ADPKD.

Disulfide bond disrupting agents activate the unfolded protein response in EGFR- and HER2-positive breast tumor cells.

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and "Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance.

CUB domain-containing protein 1 and the epidermal growth factor receptor cooperate to induce cell detachment.

BACKGROUND: While localized malignancies often respond to available therapies, most disseminated cancers are refractory. Novel approaches, therefore, are needed for the treatment of metastatic disease. CUB domain-containing protein1 (CDCP1) plays an important role in metastasis and drug resistance; the mechanism however, is poorly understood. METHODS: Breast cancer cell lines were engineered to stably express EGFR, CDCP1 or phosphorylation site mutants of CDCP1. These cell lines were used for immunoblot analysis or affinity purification followed by immunoblot analysis to assess protein phosphorylation and/or protein complex formation with CDCP1. Kinase activity was evaluated using phosphorylation site-specific antibodies and immunoblot analysis in in vitro kinase assays. Protein band excision and mass spectrometry was utilized to further identify proteins complexed with CDCP1 or ΔCDCP1, which is a mimetic of the cleaved form of CDCP1. Cell detachment was assessed using cell counting. RESULTS: This paper reports that CDCP1 forms ternary protein complexes with Src and EGFR, facilitating Src activation and Src-dependent EGFR transactivation. Importantly, we have discovered that a class of compounds termed Disulfide bond Disrupting Agents (DDAs) blocks CDCP1/EGFR/Src ternary complex formation and downstream signaling. CDCP1 and EGFR cooperate to induce detachment of breast cancer cells from the substratum and to disrupt adherens junctions. Analysis of CDCP1-containing complexes using proteomics techniques reveals that CDCP1 associates with several proteins involved in cell adhesion, including adherens junction and desmosomal cadherins, and cytoskeletal elements. CONCLUSIONS: Together, these results suggest that CDCP1 may facilitate loss of adhesion by promoting activation of EGFR and Src at sites of cell-cell and cell-substratum contact.