Mitochondrial complex II in intestinal epithelial cells regulates T cell-mediated immunopathology.

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.

Ubiquitin-specific proteases as therapeutic targets for the treatment of breast cancer.

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.

A molecularly engineered lectin destroys EGFR and inhibits the growth of non-small cell lung cancer.

Survival rates for non-small cell lung cancer (NSCLC) remain low despite the advent of novel therapeutics. Tyrosine kinase inhibitors (TKIs) targeting mutant epidermal growth factor receptor (EGFR) in NSCLC have significantly improved mortality but are plagued with challenges--they can only be used in the small fraction of patients who have susceptible driver mutations, and resistance inevitably develops. Aberrant glycosylation on the surface of cancer cells is an attractive therapeutic target as these abnormal glycosylation patterns are typically specific to cancer cells and are not present on healthy cells. H84T BanLec (H84T), a lectin previously engineered by our group to separate its antiviral activity from its mitogenicity, exhibits precision binding of high mannose, an abnormal glycan present on the surface of many cancer cells, including NSCLC. Here, we show that H84T binds to and inhibits the growth of diverse NSCLC cell lines by inducing lysosomal degradation of EGFR and leading to cancer cell death through autophagy. This is a mechanism distinct from EGFR TKIs and is independent of EGFR mutation status; H84T inhibited proliferation of both cell lines expressing wild type EGFR and those expressing mutant EGFR that is resistant to all TKIs. Further, H84T binds strongly to multiple and diverse clinical samples of both pulmonary adenocarcinoma and squamous cell carcinoma. H84T is thus a promising potential therapeutic in NSCLC, with the ability to circumvent the challenges currently faced by EGFR TKIs.

CCN6 (WISP3) decreases ZEB1-mediated EMT and invasion by attenuation of IGF-1 receptor signaling in breast cancer.

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.

Activation of the Unfolded Protein Response via Inhibition of Protein Disulfide Isomerase Decreases the Capacity for DNA Repair to Sensitize Glioblastoma to Radiotherapy.

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.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2923/F1.large.jpg.

Imaging Proteolytic Activities in Mouse Models of Cancer.

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 Promotes Survival in Human Pancreatic Cancer and Its Inhibition Suppresses Pancreatic Ductal Adenocarcinoma In Vivo Tumor Growth.

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.

New rationales for using TGFbeta inhibitors in radiotherapy.

PURPOSE: 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. RESULTS: 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. CONCLUSIONS: 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.

Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma.

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.

Three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells.

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.

Emerging potential of therapeutic targeting of ubiquitin-specific proteases in the treatment of cancer.

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.

Usp5 links suppression of p53 and FAS levels in melanoma to the BRAF pathway.

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.

Chemical derivatives of a small molecule deubiquitinase inhibitor have antiviral activity against several RNA viruses.

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.

On how CCN6 suppresses breast cancer growth and invasion.

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.

CCN6 knockdown disrupts acinar organization of breast cells in three-dimensional cultures through up-regulation of type III TGF-ß receptor.

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-ß receptor (TßRIII 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 TßRIII 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 TßRIII 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 TßRIII 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 TßRIII in maintaining acinar organization in the breast.

CCN6 modulates BMP signaling via the Smad-independent TAK1/p38 pathway, acting to suppress metastasis of breast cancer.

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-ß 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-ß 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.

TGFß1 inhibition increases the radiosensitivity of breast cancer cells in vitro and promotes tumor control by radiation in vivo.

PURPOSE: To determine whether inhibition of TGFß signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: TGFß-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 TGFß 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 TGFß signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFß neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. RESULTS: Human breast cancer cell lines pretreated with TGFß small molecule inhibitor were radiosensitized, irrespective of sensitivity to TGFß 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 TGFß type I receptor kinase inhibitor. Moreover, TGFß 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 TGFß 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.