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Figures and Tables for supplementary data. Supplementary Table 1: Primers used for qPCR analysis of PDIA1, GRP78, RAD51 and GAPDH gene expression; Supplementary Table 2: Antibodies used for western blot and inmmunocytochemistry studies; Supplementary Figure 1: PDIA1 shRNA downregulates RAD51; Supplementary Figure 2: PDI KD leads to reduced RAD51 stability in GBM cells; Supplementary Figure 3: Tunicamycin treatment leads to reduced RAD51 stability in GBM cells; Supplementary Figure 4: Silencing PDIA1 sensitizes GBM cells

Patients with glioblastoma multiforme (GBM) survive on average 12 to 14 months after diagnosis despite surgical resection followed by radiotheraphy and temozolomide therapy. Intrinsic or acquired resistance to chemo- and radiotherapy is common and contributes to a high rate of recurrence. To investigate the therapeutic potential of protein disulfide isomerase (PDI) as a target to overcome resistance to chemoradiation, we developed a GBM tumor model wherein conditional genetic ablation of prolyl 4-hydroxylase subunit beta (P4HB), the gene that encodes PDI, can be accomplished. Loss of PDI expression induced the unfolded protein response (UPR) and decreased cell survival in two independent GBM models. Nascent RNA Bru-seq analysis of PDI-depleted cells revealed a decrease in transcription of genes involved in DNA repair and cell-cycle regulation. Activation of the UPR also led to a robust decrease in RAD51 protein expression as a result of its ubiquitination-mediated proteosomal degradation. Clonogenic survival assays demonstrated enhanced killing of GBM cells in response to a combination of PDI knockdown and ionizing radiation (IR) compared with either modality alone, which correlated with a decreased capacity to repair IR-induced DNA damage. Synergistic tumor control was also observed with the combination of PDI inhibition and IR in a mouse xenograft model compared with either single agent alone. These findings provide a strong rationale for the development of PDI inhibitors and their use in combination with DNA damage-inducing, standard-of-care therapies such as IR.Significance:These findings identify PDIA1 as a therapeutic target in GBM by demonstrating efficacy of its inhibition in combination with radiotherapy through a novel mechanism involving downregulation of DNA repair genes.

PDF file, 90K, Co-localization of CCN6 and BMP4 in clinical samples of breast cancer.

PDF file, 12K, CCN6 overexpression significantly decreased tumor growth in vivo.

PDF file, 91K.

PDF file, 98K, List of TGF-B superfamily genes affected by CCN6 knockdown in HME cells measured by real time PCR.

PDF file, 121K, Confocal images and immunoblot analysis of HME CCN6 KD and control cells grown in 3D for 15 days. CCN6 KD led to disruption of acinar organization and decreased expression of E-cadherin protein. β-Actin was used as loading control.

PDF file, 330K, A. rhCCN6 protein rescues the upregulation of P-p38 and the invasive phenotype due to CCN6 KD in HME cells. B. p38 kinase activity inhibitor SB203580 reversed the invasive phenotype of CCN6 KD HME cells. C. Inhibition of p38 kinase by 20 �M SB203850 or 10 �M SB202190 is sufficient to reduce invasion of SUM149 IBC cell line and aggressive MDA-MB-231 breast cancer cells.

PDF file, 100K, A. Effect of CCN6 KD on P-Smad1, P-Smad5 and total proteins. CCN6 KD increased P-Smad1 while it downregulated P-Smad5. B. Immunoblot showing effective Smad1 siRNA inhibition in HME CCN6 KD and controls. β-Actin was used as loading control. Invasion assays show that Smad1 siRNA has not significant effect on invasion of CCN6 KD or control cells.

Intestinal epithelial cell (IEC) damage by T cells contributes to graft-versus-host disease, inflammatory bowel disease and immune checkpoint blockade-mediated colitis. But little is known about the target cell-intrinsic features that affect disease severity. Here we identified disruption of oxidative phosphorylation and an increase in succinate levels in the IECs from several distinct in vivo models of T cell-mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses, identified disruption of IEC-intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations. The relevance of IEC-intrinsic SDHA in mediating disease severity was confirmed by complementary chemical and genetic experimental approaches and validated in human clinical samples. These data identify a critical role for the alteration of the IEC-specific mitochondrial complex II component SDHA in the regulation of the severity of T cell-mediated intestinal diseases. Intestinal epithelial cell (IEC) damage by T cells contributes to alloimmune, autoimmune and iatrogenic diseases such as graft-versus-host and inflammatory bowel disease. Here the authors identify a critical role for the alteration of the IEC-specific mitochondrial complex II component succinate dehydrogenase A in the regulation of the severity of T cell-mediated intestinal diseases.

Patients with glioblastoma multiforme (GBM) survive on average 12 to 14 months after diagnosis despite surgical resection followed by radiotheraphy and temozolomide therapy. Intrinsic or acquired resistance to chemo- and radiotherapy is common and contributes to a high rate of recurrence. To investigate the therapeutic potential of protein disulfide isomerase (PDI) as a target to overcome resistance to chemoradiation, we developed a GBM tumor model wherein conditional genetic ablation of prolyl 4-hydroxylase subunit beta (P4HB), the gene that encodes PDI, can be accomplished. Loss of PDI expression induced the unfolded protein response (UPR) and decreased cell survival in two independent GBM models. Nascent RNA Bru-seq analysis of PDI-depleted cells revealed a decrease in transcription of genes involved in DNA repair and cell-cycle regulation. Activation of the UPR also led to a robust decrease in RAD51 protein expression as a result of its ubiquitination-mediated proteosomal degradation. Clonogenic survival assays demonstrated enhanced killing of GBM cells in response to a combination of PDI knockdown and ionizing radiation (IR) compared with either modality alone, which correlated with a decreased capacity to repair IR-induced DNA damage. Synergistic tumor control was also observed with the combination of PDI inhibition and IR in a mouse xenograft model compared with either single agent alone. These findings provide a strong rationale for the development of PDI inhibitors and their use in combination with DNA damage-inducing, standard-of-care therapies such as IR. Significance: These findings identify PDIA1 as a therapeutic target in GBM by demonstrating efficacy of its inhibition in combination with radiotherapy through a novel mechanism involving downregulation of DNA repair genes.

SummaryIntestinal epithelial cell (IEC) damage by T cells contributes to alloimmune, autoimmune and iatrogenic diseases such as graft-versus-host disease (GVHD), inflammatory bowel disease (IBD) and immune checkpoint blockade (ICB) mediated colitis, respectively. Despite significant advances in understanding the aberrant biology of T cells in these diseases, little is known about how the fundamental biological processes of the target IECs influence the disease severity. Here, through analyses of metabolic pathways of IECs, we identified disruption of oxidative phosphorylation without a concomitant change in glycolysis and an increase in succinate levels in several distinct in vivo models of T cell mediated gastrointestinal damage such as GVHD, IBD and ICB mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses identified a critical role for IEC intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations that contributed to the severity of intestinal damage. The critical mechanistic role of IEC intrinsic SDHA was confirmed by complementary chemical and genetic reduction of SDHA and with IEC specific deletion of SDHA. Further in vitro and in vivo mechanistic studies demonstrated that loss of SDHA in IECs was mediated by the perforin-granzyme from the T cells. The loss of SDHA was also validated in human clinical samples. These data identify a critical role for the alteration of the IEC specific mitochondrial complex II component SDHA in the regulation of the severity of T cell mediated intestinal diseases.

Proteases are "protein-cleaving" enzymes, which, in addition to their non-specific degrading function, also catalyze the highly specific and regulated process of proteolytic processing, thus regulating multiple biological functions. Alterations in proteolytic activity occur during pathological conditions such as cancer. One of the major deregulated classes of proteases in cancer is caspases, the proteolytic initiators and mediators of the apoptotic machinery. The ability to image apoptosis noninvasively in living cells and animal models of cancer can not only provide new insight into the biological basis of the disease but can also be used as a quantitative tool to screen and evaluate novel therapeutic strategies. Optical molecular imaging such as bioluminescence-based genetically engineered biosensors has been developed in our laboratory and exploited to study protease activity in animal models with a high signal to noise. Using the circularly permuted form of firefly luciferase, we have developed a reporter for Caspase 3/7, referred to as Caspase 3/7 GloSensor. Here, we discuss the use of the Caspase 3/7 GloSensor for imaging apoptotic activity in mouse xenografts and genetically engineered mouse models of cancer and present the potential of this powerful platform technology to image the proteolytic activity of numerous other proteases.

Usp9x has emerged as a potential therapeutic target in some hematologic malignancies and a broad range of solid tumors including brain, breast, and prostate. To examine Usp9x tumorigenicity and consequence of Usp9x inhibition in human pancreatic tumor models, we carried out gain- and loss-of-function studies using established human pancreatic tumor cell lines (PANC1 and MIAPACA2) and four spontaneously immortalized human pancreatic patient-derived tumor (PDX) cell lines. The effect of Usp9x activity inhibition by small molecule deubiquitinase inhibitor G9 was assessed in 2D and 3D culture, and its efficacy was tested in human tumor xenografts. Overexpression of Usp9x increased 3D growth and invasion in PANC1 cells and up-regulated the expression of known Usp9x substrates Mcl-1 and ITCH. Usp9x inhibition by shRNA-knockdown or by G9 treatment reduced 3D colony formation in PANC1 and PDX cell lines, induced rapid apoptosis in MIAPACA2 cells, and associated with reduced Mcl-1 and ITCH protein levels. Although G9 treatment reduced human MIAPACA2 tumor burden in vivo, in mouse pancreatic cancer cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, Usp9x inhibition increased and sustained the 3D colony growth and showed no significant effect on tumor growth in 8041-xenografts. Thus, Usp9x inhibition may be therapeutically active in human PDAC, but this activity was not predicted from studies of genetically engineered mouse pancreatic tumor models.

ETS transcription factors are commonly deregulated in cancer by chromosomal translocation, overexpression or post-translational modification to induce gene expression programs essential in tumorigenicity. Targeted destruction of these proteins may have therapeutic impact. Here we report that Ets-1 destruction is regulated by the deubiquitinating enzyme, Usp9x, and has major impact on the tumorigenic program of metastatic melanoma. Ets-1 deubiquitination blocks its proteasomal destruction and enhances tumorigenicity, which could be reversed by Usp9x knockdown or inhibition. Usp9x and Ets-1 levels are coincidently elevated in melanoma with highest levels detected in metastatic tumours versus normal skin or benign skin lesions. Notably, Ets-1 is induced by BRAF or MEK kinase inhibition, resulting in increased NRAS expression, which could be blocked by inactivation of Usp9x and therapeutic combination of Usp9x and MEK inhibitor fully suppressed melanoma growth. Thus, Usp9x modulates the Ets-1/NRAS regulatory network and may have biologic and therapeutic implications., Usp9x is a deubiquitinating enzyme with altered expression in melanoma; however its functional contribution in this context is not clear. Here the authors show that Usp9x regulates the stability of the transcription factor Ets-1 that in turn impacts metastatic melanoma through increased expression of NRAS.

Living cells communicate with their microenvironment and exchange information through signaling pathways in order to carry out most biological processes. The CCN family of proteins has the ability to coordinate the extracellular and intracellular signaling pathways and epithelial-stromal cross-talks. CCN proteins have been shown to play roles in multiple processes including cancer, either as tumor suppressors or oncogenes. Particularly, loss of CCN6 expression has been reported in highly aggressive breast cancer types, especially in inflammatory breast cancer and breast cancer with axillary lymph node metastasis. Recent findings can better explain the biological relevance of CCN6 as a tumor suppressor protein in breast tumorigenesis. CCN6 loss triggers the process of epithelial to mesenchymal transition (EMT), which converts epithelial cells into migratory and invasive mesenchymal-like cells at least in part through modulation of IGF-1 receptor signaling pathway. Emerging data support the hypothesis that CCN6 also exerts growth factor independent functions, especially related to cell survival and anoikis resistance. Thus, our work provides new insights into the functions and mechanisms of tumor suppression exerted by CCN6 in the breast.

Purpose – This paper aims to provide an overview of legal reference sources available from ProQuest, specifically Legislative Insight and Congressional, with a focus on their scope and usability for particular types of legal reference queries. Design/methodology/approach – ProQuest resources useful for legal research were located, evaluated and discussed. Findings – ProQuest legal resources are not as prominently displayed as many of the vendor’s other subject-specific offerings, but once located, several of them can be used collaboratively to fully research a single legal issue from inception to practice and enforcement. Originality/value – The examination of the resources in this study will provide academic librarians with an understanding of which ProQuest resources should be used in collaboration with each other to answer general legal enquiries as well as in-depth, specific statutory research.

The first reports that ionizing radiation (IR) induces rapid and persistent activation of transforming growth factor beta1 (TGFbeta) were nearly two decades ago. Subsequent studies have shown that TGFbeta is a major mediator of cellular and tissue responses to IR and have revealed novel facets of its complex biology.We and others have recently shown that inhibition of production or signaling of TGFbeta in epithelial cells modulates radiosensitivity and impedes activation of the DNA damage response program. The primary transducer of cellular response to DNA damage caused by ionizing radiation is the nuclear protein kinase ataxia telangiectasia mutated, whose activity is severely compromised when TGFbeta is inhibited. Thus, in conjunction, with its well-recognized contribution to normal tissue fibrosis, the role of TGFbeta in the genotoxic stress program provides a previously unsuspected avenue to modulate radiotherapy.We hypothesize that identification of the circumstances and tumors in which TGFbeta manipulation enhances tumor cell radiosensitivity, while protecting normal tissues, could significantly increase therapeutic index.

The ubiquitin–proteasome system (UPS) has emerged as a therapeutic focus and target for the treatment of cancer. The most clinically successful UPS-active agents (bortezomib and lenalidomide) are limited in application to hematologic malignancies, with only marginal efficacy in solid tumors. Inhibition of specific ubiquitin E3 ligases has also emerged as a valid therapeutic strategy, and many targets are currently being investigated. Another emerging and promising approach in regulation of the UPS involves targeting deubiquitinases (DUB). The DUBs comprise a relatively small group of proteins, most with cysteine protease activity that target several key proteins involved in regulation of tumorigenesis, apoptosis, senescence, and autophagy. Through their multiple contacts with ubiquitinated protein substrates involved in these pathways, DUBs provide an untapped means of modulating many important regulatory proteins that support oncogenic transformation and progression. Ubiquitin-specific proteases (USP) are one class of DUBs that have drawn special attention as cancer targets, as many are differentially expressed or activated in tumors or their microenvironment, making them ideal candidates for drug development. This review attempts to summarize the USPs implicated in different cancers, the current status of USP inhibitor–mediated pharmacologic intervention, and future prospects for USP inhibitors to treat diverse cancers. Cancer Res; 74(18); 4955–66. ©2014 AACR.

Usp5 is a deubiquitinase (DUB) previously shown to regulate unanchored poly-ubiquitin (Ub) chains, p53 transcriptional activity and double-strand DNA repair. In BRAF mutant melanoma cells, Usp5 activity was suppressed by BRAF inhibitor (vemurafenib) in sensitive but not in acquired or intrinsically resistant cells. Usp5 knockdown overcame acquired vemurafenib resistance and sensitized BRAF and NRAS mutant melanoma cells to apoptosis initiated by MEK inhibitor, cytokines or DNA-damaging agents. Knockdown and overexpression studies demonstrated that Usp5 regulates p53 (and p73) levels and alters cell growth and cell cycle distribution associated with p21 induction. Usp5 also regulates the intrinsic apoptotic pathway by modulating p53-dependent FAS expression. A small molecule DUB inhibitor (EOAI3402143) phenocopied the FAS induction and apoptotic sensitization of Usp5 knockdown and fully blocked melanoma tumor growth in mice. Overall, our results demonstrate that BRAF activates Usp5 to suppress cell cycle checkpoint control and apoptosis by blocking p53 and FAS induction; all of which can be restored by small molecule-mediated Usp5 inhibition. These results suggest that Usp5 inhibition can provide an alternate approach in recovery of diminished p53 (or p73) function in melanoma and can add to the targeted therapies already used in the treatment of melanoma.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior(1). However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system(2,3). Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D(4). 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved(3). One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D(6,7). Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Most antiviral treatment options target the invading pathogen and unavoidably encounter loss of efficacy as the pathogen mutates to overcome replication restrictions. A good strategy for circumventing drug resistance, or for pathogens without treatment options, is to target host cell proteins that are utilized by viruses during infection. The small molecule WP1130 is a selective deubiquitinase inhibitor shown previously to successfully reduce replication of noroviruses and some other RNA viruses. In this study, we screened a library of 31 small molecule derivatives of WP1130 to identify compounds that retained the broad-spectrum antiviral activity of the parent compound in vitro but exhibited improved drug-like properties, particularly increased aqueous solubility. Seventeen compounds significantly reduced murine norovirus infection in murine macrophage RAW 264.7 cells, with four causing decreases in viral titers that were similar or slightly better than WP1130 (1.9 to 2.6 log scale). Antiviral activity was observed following pre-treatment and up to 1 hour postinfection in RAW 264.7 cells as well as in primary bone marrow-derived macrophages. Treatment of the human norovirus replicon system cell line with the same four compounds also decreased levels of Norwalk virus RNA. No significant cytotoxicity was observed at the working concentration of 5 µM for all compounds tested. In addition, the WP1130 derivatives maintained their broad-spectrum antiviral activity against other RNA viruses, Sindbis virus, LaCrosse virus, encephalomyocarditis virus, and Tulane virus. Thus, altering structural characteristics of WP1130 can maintain effective broad-spectrum antiviral activity while increasing aqueous solubility.

Transcription factors are frequently deregulated in cancer cells and may be good therapeutic targets, but few successful targeting strategies have been reported. Deubiquitinases (DUBs) are specialized enzymes that regulate the ubiquitin (Ub) content on many proteins and DUB expression and activity are elevated in a number of cancers where they can act to alter tumor suppressor and/or oncoprotein levels. We previously described Usp9x activity and expression in melanoma; here we sought to investigate its role in primary melanoma and metastatic disease. Usp9x was upregulated in tumor cells compared to normal melanocytes and Usp9x expression and activity were found to be essential for 3D growth and melanoma tumor expansion in vivo. We defined the Usp9x ubiquitinated protein landscape and demonstrate that Usp9x regulates Ets-1, a cancer-promoting transcription factor. Usp9x binds, deubiquitinates and thereby stabilizes Ets-1 protein, and primary tissue and tumor analysis demonstrated elevated and coincident Usp9x/Ets-1 protein expression in melanoma compared to normal skin or benign nevi. Usp9x knockdown or Usp9x inhibition with small molecule G9 reduced Ets-1 protein levels and blocked tumor growth in vitro and in vivo. Conversely, Usp9x overexpression in melanoma cells increased Ets-1 protein levels and enhanced 3D tumor growth in vitro and in vivo, which were all reversible by treatment with G9. We conclude that Usp9x is essential for Ets-1 protein stability and may be therapeutically exploited with small molecule Usp9x inhibitors to reduce Ets-1-dependent gene expression and tumorigenicity. Citation Format: Harish Potu, Luke F. Peterson Peterson, Malathi Kandarpa Kandarpa, Anupama Pal Pal, Hanshi Sun Sun, Paul W. Harms Harms, Peter C. Hollenhorst Hollenhorst, Ugur Eskiocak Eskiocak, Moshe Talpaz Talpaz, Nicholas J. Donato Donato. Deubiquitinase Usp9x controls tumorigenicity through regulation of the Ets-1 transcription factor in melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2881.

CCN6 (WISP3) is an extracellular matrix protein that exerts tumor suppressive functions in breast cancer, where its decreased expression is a feature of advanced disease. However, neither its role nor mechanism of action in breast cancer metastasis has been established. Bone morphogenetic proteins (BMPs), which constitute ligands of the TGF-β superfamily, are multifunctional cytokines that induce epithelial–mesenchymal transition, cell invasion, and metastasis. In this study, we identify a CCN6-BMP4-TAK1 kinase signaling pathway that controls the ability of the p38 MAP kinase to regulate acinar morphogenesis and invasion of breast cells. ShRNA-mediated attenuation of CCN6 in human mammary epithelial cells led to BMP4 upregulation as a major response to exposure to the TGF-β superfamily. CCN6 attenuation also induced BMP4-mediated activation of the Smad-independent TAK1 and p38 kinases. Conversely, ectopic expression of CCN6 in breast cancer cells antagonized BMP4-mediated TAK1/p38 activation and invasive capacity, both by binding BMP4 protein as well as decreasing BMP4 protein levels. Effects on BMP4 and p38 were confirmed in vivo where they correlated with decreased metastasis. In clinical specimens, we found that CCN6 expression was inversely associated with BMP4 and phospho-p38 levels in 69% of invasive breast carcinomas examined, consistent with the functional results. Together our findings identify a novel modifier pathway through which CCN6 acts to limit breast cancer invasion and metastasis. Cancer Res; 72(18); 4818–28. ©2012 AACR.

Purpose: To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. Experimental Design: TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. Results: Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFβ type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. Conclusion: These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy. Clin Cancer Res; 17(21); 6754–65. ©2011 AACR.

During progression of breast cancer, CCN6 protein exerts tumor inhibitory functions. CCN6 is a secreted protein that modulates the insulin-like growth factor-1 (IGF-1) signaling pathway. Knockdown of CCN6 in benign mammary epithelial cells triggers an epithelial to mesenchymal transition (EMT), with upregulation of the transcription factor ZEB1/δEF1. How CCN6 regulates ZEB1 expression is unknown. We hypothesized that CCN6 might regulate ZEB1, EMT and breast cancer invasion by modulating IGF-1 signaling. Exogenously added human recombinant CCN6 protein was sufficient to downregulate ZEB1 mRNA and protein levels in CCN6-deficient (CCN6 KD) HME cells and MDA-MB-231 breast cancer cells. Recombinant CCN6 protein decreased invasion of CCN6 KD cells compared with controls. We discovered that knockdown of CCN6 induced IGF-1 secretion in HME cells cultivated in serum-free medium to higher concentrations than found in MDA-MB-231 cells. Treatment with recombinant CCN6 protein was sufficient to decrease IGF-1 protein and mRNA to control levels, rescuing the effect of CCN6 knockdown. Specific inhibition of IGF-1 receptors using the pharmacological inhibitor NVP-AE541 or short hairpin shRNAs revealed that ZEB1 upregulation due to knockdown of CCN6 requires activation of IGF-1 receptor signaling. Recombinant CCN6 blunted IGF-1-induced ZEB1 upregulation in MDA-MB-231 cells. Our data define a pathway in which CCN6 attenuates IGF-1 signaling to decrease ZEB1 expression and invasion in breast cancer. These results suggest that CCN6 could be a target to prevent or halt breast cancer invasion. Fil: Lorenzatti, Guadalupe. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Huang, Wei. University Of Michigan. Department Of Pathology; Estados Unidos Fil: Pal, Anupama. University Of Michigan. Department Of Pathology; Estados Unidos Fil: Cabanillas, Ana Maria de Los A.. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Kleer, Celina G.. University Of Michigan. Department Of Pathology; Estados Unidos

Living cells communicate with the surrounding microenvironment and exchange information through complex signaling pathways in order to carry out most homeostatic biological processes. Matricellular proteins, which include the CCN family of proteins, have the ability to coordinate the extracellular and intracellular signaling pathways and thus modulate the cross-talk between the microenvironment, epithelial and mesenchymal cells. Our laboratory has been devoted to understand the functions of CCN6 in breast tumorigenesis. Loss of CCN6 has been recognized as a genetic determinant for inflammatory breast cancer, a highly aggressive and metastatic form of breast cancer. Further investigations have shown that CCN6 has suppressive roles in the growth and invasion of inflammatory and non-inflammatory breast cancer cells. In recent years, we have made two important discoveries towards understanding CCN6’s mechanism of function. We found that CCN6 loss triggers the process of epithelial to mesenchymal transition (EMT), which convert epithelial cells into migratory mesenchymal cells and thus drive breast cancers with metastatic ability. We have also found that CCN6 downregulation enhances the proliferation and survival effects of extracellular growth factors on mammary epithelial cells. Thus, our work provides new insights into the mechanism of tumor suppression exerted by CCN6 in the breast.

Usp9x regulates tumor cell survival and responsiveness to targeted-, chemo- and radio-therapy in a broad number of tumors through effects on multiple proteins. The role of Usp9x in human pancreatic cancer is largely unknown, although a recent study demonstrated that Usp9x co-operated in promoting KrasG12D tumorigenesis in mice. To examine Usp9x tumorigenicity and therapeutic potential of Usp9x inhibition in human pancreatic cancer vs. mouse pancreatic tumor models we developed 3D cultures of cell lines established from constitutive (8041) and doxycycline-inducible (4668) KrasG12D/Tp53R172H mouse pancreatic tumors, established human cell lines (BxPC3, PANC1 and MIAPACA2) and spontaneously immortalized human pancreatic patient tumor derived cell lines (UM2, UM6, UM16 and UM76). The effect of Usp9x knockdown (KD), overexpression (OE) or Usp9x activity inhibition by our small molecule deubiquitinase inhibitor EOAI3402143 (G9) were assessed by growth in 2D and 3D culture and anti-tumor efficacy studies in mouse (8041) and human (MIAPACA2) xenograft models. Usp9x KD in constitutive KrasG12D/Tp53R172H 8041 cells led to a 3-fold increase in the number of colonies formed in 3D and Usp9x KD sustained 3D colony growth even after withdrawal of mutant Kras support in KrasG12D/Tp53R172H inducible 4668 cells. Usp9x-OE in 8041 cells decreased 3D colony growth by 2.5-fold. These results contrast with outcomes in human pancreatic tumors where Usp9x KD induced either rapid apoptosis (in MIAPACA2) or reduced 3D colony formation by >50% (in PANC1). Usp9x-OE in PANC1 cells enhanced 3D colony growth (2-fold) and increased invasion activity in Boyden chambers (3.5-fold). Mechanistically, we found differential effects of Usp9x on mutant Kras protein expression levels in mouse vs human cells. In vivo G9 treatment (15 mg/kg, every other day) of mouse 8041 xenografts showed a statistically insignificant trend towards tumor growth inhibition but effectively inhibited MIAPACA2 tumor growth in mice. Usp9x KD in human pancreatic patient tumor cell lines inhibited 3D colony growth by >75% and this activity was phenocopied by G9 with nM efficacy. We conclude that Usp9x acts as a tumor promoter in established human pancreatic cancer and a tumor suppressor in murine cells from genetically engineered pancreatic tumors, possibly through differential regulation of mutant Kras. Overall, these results suggest that Usp9x may be a novel therapeutic target in pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Anupama Pal, Marina Pasca Di Magliano, Diane Simeone, Luke Peterson, Harish Potu, Moshe Talpaz, Nicholas Donato. Usp9x as a novel therapeutic target in human pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2015-1748

Melanoma is an aggressive skin cancer characterized by gene mutations, high metastatic potential and few clinically effective systemic therapies. BRAF, MEK1 kinase inhibitors and CTLA4 antagonist have improved patient survival, but only a subset of patients respond or derived long-lasting clinical benefit, illustrating the need for discovery of additional targets and pathways that may be inhibited with small molecules. Given the clinical impact of inhibitors of the ubiquitin-proteasome system in other cancers, we explored the potential for aberrant deubiquitinase (DUB) activity to play a role in the tumorigenic properties of melanoma. We also assessed whether specific DUBs play a role in apoptotic responsiveness and resistance to kinase inhibitors. Initially, we noted that the activity of several DUBs, including Usp9x and Usp5, was elevated in melanoma compared to normal melanocytes. Further, Usp9x and Usp5 expression and activity were up-regulated by expression of BRAFV600E in 293T cells and kinase inhibition partially suppressed both Usp9x and Usp5 activity in vemurafenib sensitive, but not resistant cells. Usp9x knockdown (KD) in BRAF mutant or wild-type melanoma did not affect 2-dimensional cell growth or survival but significantly amplified the apoptotic activity of BRAF or MEK1 inhibitors, respectively. Usp5 KD in melanoma suppressed cell growth by reinforced the S/G2-M checkpoint, enhanced extrinsic caspase activation through modulation of p53 and FAS levels and amplified the apoptotic activity of kinase inhibitors (and other chemotherapeutic agents) and overcame vemurafenib resistance in A375R cells. To further assess the clinical relevance of activated DUBs in melanoma, primary human melanoma explants characterized for their metastatic efficiency in patients and NSG mice demonstrated that Usp9x and Usp5 expression and activity were elevated in efficient metastasizers, but only a subset of inefficient metastasizers, suggesting a potential role for DUBs in the regulation of metastatic potential. To investigate that possibly, control and Usp9x KD cells were grown in Matrigel or suspension culture and colony growth and survival, as well as Usp9x activity were examined. Usp9x KD significantly suppressed melanoma colony growth and fully reduced survival of highly metastatic SK-Mel147 cells in 3D culture. Growth in 3D conditions resulted in activation of Usp9x, but also increased their sensitivity (IC50 Citation Format: Harish Potu, Anupama Pal, Hanshi Sun, Luke Peterson, Moshe Talpaz, Monique Verhaegen, Juxiang Cao, Ugur Eskiocak, Sean Morrison, Nicholas J. Donato. Deubiquitinases Usp9x and Usp5 control tumorigenicity and apoptotic responsiveness in malignant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4435. doi:10.1158/1538-7445.AM2014-4435

Deubiquitinase Usp9x has been shown to promote tumor cell survival and resistance to chemo- and radiotherapy through effects on the Mcl-1 pro-survival protein. It is predicted that a small molecule Usp9x inhibitor could have a major impact in cancer therapy. However, recent data has raised caution about therapeutic targeting of Usp9X in cancer. In a study to identify genes that reduce the latency of pancreatic tumor formation in a mutant Kras model, Pérez-Mancera et al. 2012 reported that the Usp9x locus was frequently inactivated in pancreatic cancer cells, resulting in protection from anoikis and enhanced transformation. In order to clarify the role of Usp9X in pancreatic cancer, we used a newly created cell line 4668 established from mouse pancreatic tumors with doxycycline (DOX) inducible expression of KrasG12D and Tp53R172H. Stable Usp9x knockdown (KD) in 4668 cells had no effect on their proliferation or phenotype in 2D cultures. However, withdrawal of DOX from DOX-treated Usp9x KD 4668 cells resulted in 4-fold increase in colony formation, unlike DOX depleted control KD 4668 cells which undergo apoptosis resulting in colony disintegration. DOX treated control KD and Usp9x KD 4668 cells did not show any difference in basal 3D colony formation. These results suggest that Usp9x loss in the absence of continuous mutant Kras/p53 expression is sufficient to sustain 3D cancer cell growth, confirming a tumor suppressor role for Usp9x in a Kras mouse model of pancreatic cancer. To determine if Usp9x also plays a tumor suppressor role in human pancreatic tumors, we did Usp9x KD in three human pancreatic cancer cell lines with varied Kras mutational status; BxPC3 (wild type), PANC1 (heterozygous) and MIA-PACA2 (homozygous) which expressed high basal levels of Usp9x. MIA-PACA2-Usp9x KD cells undergo apoptosis and caspase activation. In contrast, BxPC3-Usp9x KD cells showed no effect on growth in 2D or in 3D culture. However, PANC1-Usp9x KD cells showed more rapid (38% p=0.004) 2D growth, but 3D colony formation was consistently reduced by >50%, (p=0.03). These results suggest that the effect of Usp9x loss in established human pancreatic tumor cells is distinct from that detected in a murine pancreatic tumor model. Distinctions may be partially dependent on their Kras mutational status or Usp9x substrate utilization in human vs. murine cells. To further assess the clinical relevance of our observations, Usp9x KD was evaluated in four recently established, low passage number human pancreatic patient tumor cell lines (UM2, UM6, UM16, UM76). Usp9x KD did not affect 2D growth but fully suppressed 3D colony growth. This activity was phenocopied by our small molecule Usp9x inhibitor, EOAI3402143 (G9), with nM efficacy. Together, these results suggest that possible species distinctions exist in the tumorigenic functions of Usp9x and necessitate further assessment of its role in pancreatic tumor development and treatment. Citation Format: Anupama Pal, Marina Pasca di Magliano, Moshe Talpaz, Michelle Dziubinski, Diane Simeone, Nicholas J. Donato. Role of Deubiquitinase Usp9x in pancreatic cancers: Tumor promoter or tumor suppressor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4434. doi:10.1158/1538-7445.AM2014-4434

Highly metastatic Triple Negative (ER/PR and HER2 negative) breast cancer (TNBC) comprises 20% of all breast cancers but accounts for a disproportionate share of mortality due to its aggressive nature and limited therapeutic options. Thus novel metastatic breast cancer targets and inhibitors need to be defined. Usp9x is a deubiquitinase that has been shown to regulate the stability and function of many signaling, cell cycle regulatory and survival proteins and has emerged as a negative prognosticator and potential therapeutic target in several cancers. Usp9x functions in polarization and survival of tumor cells derived from various sites of origin. Additional studies have shown that Usp9x expression is required to maintain growth of glioblastoma and medulloblastomas and it suppresses the apoptotic effects of targeted-, chemo- and radio-therapy in a broad spectrum of tumors. Previous studies also demonstrated that Usp9x and Mcl-1 protein levels are highly elevated in ductal breast carcinoma when compared to normal tissue. Thus, we hypothesized that Usp9x may play a role in tumor and metastasis promoting functions in breast cancer. We first assessed Usp9x activity and total protein expression in a panel of benign (HME, MCF10A), tumorigenic (MCF7 and T47D) and highly metastatic (SUM149 and MDAMB231) human breast cell lines and found up-regulation of Usp9x enzymatic activity and total protein in tumorigenic and metastatic breast cancers as compared to benign breast cells. Increased Usp9x expression also correlated with increased levels of Mcl-1. Knockdown (KD) of Usp9x in four TNBC cell lines [MDAMB231, MDAMB436, BT549 and SUM159] triggered phenotypic apoptotic events like membrane blebbing and nuclear fragmentation, which were further confirmed by the presence of activated caspases, cleaved PARP and reduced Mcl-1 protein expression. Importantly Usp9x KD specifically induced apoptosis in metastatic breast cancer cells with a mesenchymal stem cell-like phenotype (EpCAM+/CD24-/CD44+ population of cells) and fully suppressed TNBC colony growth and acinar structures when grown on Matrigel. Usp9x KD in tumorigenic MCF7 cells resulted in growth inhibition with G2 arrest, without evidence of apoptosis, whereas Usp9x KD had no effect on the growth or survival of benign breast cells. To examine the therapeutic potential of Usp9x inhibitors in breast cancer we employed EOAI3402143 (G9), a small molecule Usp9x inhibitor developed in our laboratory with efficacy in myeloma tumor regression in mice. G9 inhibited Usp9x activity, reduced Mcl-1 levels and induced apoptosis in TNBC cells within 4h of inhibitor treatment. G9 also phenocopied the effect of Usp9x KD in 3D culture with nM activity. Collectively, our data shows that Usp9x plays a role in breast tumor growth and survival and its inhibition in tumorigenic and highly metastatic breast cancers may provide a novel therapy for the treatment of metastatic breast cancer. Citation Format: Anupama Pal, Ming Luo, Max Wicha, Moshe Talpaz, Nicholas Donato. Deubiquitinase USP9X inhibition induces apoptosis in metastatic breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2595. doi:10.1158/1538-7445.AM2014-2595

While normal cells in the human breast are organized into acinar structures, disruption of the acinar architecture is a hallmark of cancer. In a three-dimensional model of morphogenesis, we show that down-regulation of the matrix-associated tumor suppressor protein CCN6 (WNT1-inducible-signaling pathway protein 3) disrupts breast epithelial cell polarity and organization into acini through up-regulation of the type III transforming growth factor-β receptor (TβRIII or betaglycan). Down-regulation of CCN6 in benign breast cells led to loss of tissue polarity and resulted in cellular disorganization with loss of α6 integrin-rich basement membrane and the basolateral polarity protein E-cadherin. Silencing of TβRIII with shRNA and siRNA rescued the ability of breast epithelial cells to form polarized acinar structures with reduced matrix invasion and restored the correct expression of α6 integrin and E-cadherin. Conversely, CCN6 overexpression in aggressive breast cancer cells reduced TβRIII in vitro and in a xenograft model of CCN6 overexpression. The relevance of our studies to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely associated with TβRIII protein in 64%of invasive breast carcinomas. These results reveal a novel function of the matricellular protein CCN6 and establish a mechanistic link between CCN6 and TβRIII in maintaining acinar organization in the breast.

Epithelial to mesenchymal transition (EMT) is an important event during tumor invasion and metastasis. EMT is regulated by extracellular matrix components and soluble growth factors (reviewed in Nat Rev Mol Cell Biol 7:131-42, 2006). Secreted protein CCN6 is a multimodular matricellular regulatory protein, reported to be lost in 80% cases of highly aggressive inflammatory breast cancer subtype and 60% cases of all invasive breast carcinomas. Our previous studies showed that CCN6 regulates epithelial differentiation and its loss triggers EMT in human mammary epithelial cells (HME) through loss of E-cadherin by inducing Snail and Zeb1 (Am J Pathol 172(4):893-04, 2008). However the mechanism is not known. TGFβ signaling is known to cause EMT and invasion at advance stages of tumor progression by inducing Zeb1 (reviewed in J Mammary Gland Biol Neoplasia 15(2):169-90, 2010). Since, the structure of CCN6 predicts binding sites for TGFβ, we hypothesized that CCN6 loss induced EMT and invasion is mediated through deregulation of TGFβ signaling. HME cells with lentivirus mediated knockdown (KD) of CCN6 were characterized by 3 dimensional cultures. TGFβ-Smad response was examined in serum starved cells in a dose and time dependent manner. Growth, invasion, EMT and Smad-response were tested in control and CCN6 KD cells with siRNA or lentivirus mediated inhibition of TGFβ type III receptor (TβR III); and in CCN6 overexpressing human metastatic MDAMB231 cells (which have low protein levels of CCN6) in vitro and in vivo. We found that the CCN6 KD cells present a very malignant phenotype with loss of alpha 6 integrin. Associated with this, we observed an upregulaton of transcript (40 fold, *) and protein levels of TβR III, and enhanced activation of Smad2 in response to TGFβ1/TGFβ2/TGFβ3. Inhibition of upregulated TβR III blunted invasion and growth by reducing pSmad2 levels. The CCN6/TβR III double KD cells showed mesenchymal to epithelial transition with re-expression of E-cadherin and reversion of the malignant phenotype into polarized acini. Strengthening our findings CCN6 overexpression in MDAMB231 cells reduced invasion and growth by reducing pSmad2 levels. In vivo, CCN6 overexpressing mammary tumors showed dramatic decrease in tumor volume (70% at 7 weeks of injection, *, n=15 mice/group) and prolonged metastasis free survival with the primary tumors and mets showing more epithelial phenotype, unlike the EMT-like phenotype of controls. Further, treating CCN6 KD cells with 500 ng/ml of recombinant human CCN6 reduced transcript levels of TβR III while no effect was observed on control cells. Conclusively, CCN6 loss induced EMT is mediated through upregulation of TβR III. CCN6 can maintain the epithelial phenotype by regulating E-cadherin through modulation of TβR III. Thus, CCN6 emerges as a therapeutic target that can suppress tumor progression by modulating TGFβ signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 14. doi:1538-7445.AM2012-14

CCN6 is a secreted protein with tumor suppressor function in the most aggressive form of locally advanced breast cancer, inflammatory breast cancer. CCN6 was reported to be lost in 80% cases of inflammatory breast cancer and 19% cases of stage matched non inflammatory breast cancer. It was shown that CCN6 knockdown (KD) in benign breast cells triggered an epithelial to mesenchymal transition with E-cadherin downregulation (Am J Pathol 172(4):893-904, 2008) recently shown to be associated with acquisition of growth factor independent signaling (Cancer Res 70 (8):3340-3350, 2010). The molecular mechanism by which CCN6 regulates extracellular signaling to suppress breast cancer invasion is not known. As a member of the CCN family of matricellular proteins, named after connective tissue growth factor, cysteine-rich 61, and nephroblastoma overexpressed, CCN6 shares the highly conserved protein motifs of the CCN family, which in some cases have been reported to bind to bone morphogenetic protein (BMP) and transforming growth factor beta (TGFβ) (J Cell Sci 119(23):4803-4810, 2006). We hypothesized that the invasive phenotype of CCN6 KD cells may be dependent upon cytoskeletal alterations through modulation of growth factor signaling pathways like TGFβ/BMP. The invasive phenotype of lentivirus mediated control and CCN6 KD cells was assessed by growth on matrigel for 15 days following published protocol. The effect on TGFβ/BMP signaling was examined by a pathway specific PCR array for TGFβ/BMP (SA Biosciences), immunoblotting and immunocytochemistry. To ascertain the tumor suppressor effect of CCN6, we examined if recombinant human CCN6 (rhCCN6) could revert the invasive phenotype by treatment with 500 ng/ml rhCCN6 (PeproTech). Similarly small molecule inhibitors were used to inhibit activated P38 kinase (5 μM SB203580), TGFβ type I receptor I kinase (TβRI) (400 nM SB431542) and TGFβ activated kinase1 (TAK1) (0.3 μM 5Z-7-oxozeaenol). The specific binding of CCN6 with BMP4 was confirmed by co-immunoprecipitation, crosslinking, competitive binding and surface plasmon resonance. We found that CCN6 loss in benign breast cells triggers a highly invasive phenotype with loss of α6 integrin, which was rescued by treatment with rhCCN6. CCN6 loss in HME cells antagonized BMP4-mediated Smad1/5 activation and enhanced the effect of BMP4 on the non-canonical pathway to activate TAK1 and P38 through activation of TβRI. Small molecule inhibition of activated P38 or TAK1 or TβRI, or of BMP4 by blocking antibody reverted the invasive phenotype. CCN6 protein had the opposite effect, potentiation of BMP4-mediated Smad1/5 signaling by direct binding to BMP4. Conclusively, by direct binding to BMP4 CCN6 regulates BMP4 mediated Smad and alternate P38 signaling. Thus, CCN6 in the extracellular space may act as a master switch and regulator of BMP4 to suppress invasion in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2200. doi:10.1158/1538-7445.AM2011-2200

Background: During cancer progression, the transcription factor ZEB1 has been implicated in the regulation of key genes triggering the EMT (Epithelial to Mesenchymal Transition) program. We previously reported that the matricellular protein CCN6 is lost or reduced in highly aggressive breast carcinomas. Furthermore, we have identified CCN6 as a tumor suppressor in breast cancer whose down-regulation enhances IGF-1 signaling on breast epithelial cells. Recently, our lab observed that stable inhibition of CCN6 in benign mammary epithelial cells causes EMT and invasion with marked up-regulation of ZEB1. Since IGF-1 is shown to up-regulate ZEB1 expression in epithelial prostate cancer cells we hypothesize that CCN6 may regulate EMT in breast cancers by modulating IGF-1 signaling and thereby regulating ZEB1 expression. Methods: Stable CCN6-deficient HME (Human Mammary Epithelial cells)(HME-CCN6 KD) were incubated with 500 ng/mL rhCCN6 protein. Western blots (WB) for ZEB1 and vimentin, inmunofluorescence, invasion and proliferation assays were performed. ZEB1 mRNA levels were evaluated by RT-qPCR (Taqman). IGF-1 expression was analyzed by RT-qPCR and WB on MDA-MB-231 and HME-CCN6 KD (pre- and post-rhCCN6 treatment) while IGF-1R, phospho-IGF-1R, IRS-1 and phospho-IRS-1 expression were investigated by WB. Lentivirus-mediated (pLK.O1) short hairpin RNA targeting IGF1-R was performed in HME control and HME-CCN6 KD cells. WBs were done to assess expression of proteins of interest. mRNA content was evaluated by RT-qPCR. Results: CCN6 incubation of HME-CCN6 KD cells induced down-regulation of ZEB1 mRNA and protein levels as well as subcellular localization changes of the mesenchymal marker vimentin (“cap” pattern) evidenced as a decrease in vimentin expression by WB. hrCCN6 protein treatment also reduced proliferation and invasion abilities of HME-CCN6 KD cells compared to controls. In Addition, cell lines expressing low levels of CCN6 such us HME-CCN6 KD and MDA-MB-231 secreted IGF-1 in the conditioned media and showed activation of the IGF-1 signaling pathway. This phenomenon was reverted by CCN6 treatment, with reduction of both, IGF-1 protein and mRNA levels. IGF-1R down-regulation in HME-CCN6 KD cells decreased ZEB1 expression and induced the re-expression of cytokeratin-18 (epithelial marker). Conclusions: Our results suggest that CCN6 regulates ZEB1 expression and EMT in breast epithelial cells by modulating the IGF-1 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3172.

Key mediators of signaling pathways in breast cancer involve post-translational protein modification, primarily mediated through phosphorylation and ubiquitination. While previous studies focused on phosphorylation events, more recent analysis suggests that ubiquitin plays a parallel and equally important role in several signaling and cell regulatory events in breast cancer. Availability of new tools capable of sensitive detection of gene mutations and aberrant expression of genes and proteins coupled with gene-specific knockdown and silencing protocols have provided insight into the previously unexplored ubiquitin regulatory process within these tumors. Ubiquitin-specific proteases are one class of enzymes with protein deubiquitinating activity, making up the majority of protein deubiquitinating diversity within mammalian cells. Ubiquitin-specific proteases are also emerging as potential therapeutic targets in many diseases, including cancer. In this report, we summarize the involvement of this class of enzymes in breast cancer signaling and cell regulation and illustrate the potential for additional studies to define novel targets and approaches in breast cancer therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0461-3) contains supplementary material, which is available to authorized users.