Search

Supplementary Materials and Methods. Figure S1: Quantitative Real-time PCR. Figure S2: MCF7 cells were transfected with HIF-1α siRNA oligos and control siRNA oligos before exposing them to hypoxia (3% oxygen) for indicated time intervals. Figure S3 : MTT assay was performed on MCF10DCIS batch clones after 24 hours of treatments as indicated. Figure S4: (A and B) pcDNA3.1 or pcDNA3.1 HSulf-1-myc/His plasmid transfected MCF10DCIS. Figure S5: K means cluster analysis of HSulf-1 expression with hypoxia signature genes as described in supplementary materials and methods. Figure S6: Kaplan-Meier survival analysis was performed to evaluate HSulf-1 mRNA expression levels with disease-free and overall patient survival. Figure S7: Proposed model. Table S1: Primers used for cloning, ChIP and siRNA oligos. Table S2: Patient Characteristics. Table S3: Chi-square statistical analysis of HSulf-1 and CAIX expression. Table S4: Prognostic factors in breast cancer cases compared with disease-free survival. Table S5: Prognostic factors in breast cancer cases compared with overall survival. Table S6: Characteristics of clinical parameters of breast cancer and HSulf-1 mRNA.

HSulf-1 modulates the sulfation states of heparan sulfate proteoglycans critical for heparin binding growth factor signaling. In the present study, we show that HSulf-1 is transcriptionally deregulated under hypoxia in breast cancer cell lines. Knockdown of HIF-1α rescued HSulf-1 downregulation imposed by hypoxia, both at the RNA and protein levels. Chromatin immunoprecipitation with HIF-1α and HIF-2α antibodies confirmed recruitment of HIF-α proteins to the two functional hypoxia-responsive elements on the native HSulf-1 promoter. HSulf-1 depletion in breast cancer cells resulted in an increased and sustained bFGF2 (basic fibroblast growth factor) signaling and promoted cell migration and invasion under hypoxic conditions. In addition, FGFR2 (fibroblast growth factor receptor 2) depletion in HSulf-1–silenced breast cancer cells attenuated hypoxia-mediated cell invasion. Immunohistochemical analysis of 53 invasive ductal carcinomas and their autologous metastatic lesions revealed an inverse correlation for the expression of HSulf-1 to CAIX in both the primary tumors (P ≥ 0.0198) and metastatic lesions (P ≥ 0.0067), respectively, by χ2 test. Finally, HSulf-1 expression levels in breast tumors by RNA in situ hybridization showed that high HSulf-1 expression is associated with increased disease-free and overall survival (P ≥ 0.03 and P ≥ 0.0001, respectively). Collectively, these results reveal an important link between loss of HSulf-1 under hypoxic microenvironment and increased growth factor signaling, cell migration, and invasion. Cancer Res; 71(6); 2152–61. ©2011 AACR.

Supplementary Figure 1 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Table 1 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Methods from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Figure Legends 1-2 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Table 2 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Figure 2 from Regulation of HSulf-1 Expression by Variant Hepatic Nuclear Factor 1 in Ovarian Cancer

Supplementary Methods, Figures 1-7, Tables 1-6 from HSulf-1 Modulates FGF2- and Hypoxia-Mediated Migration and Invasion of Breast Cancer Cells

Metabolic alterations are increasingly recognized as important novel anti-cancer targets. Among several regulators of metabolic alterations, fructose 2,6 bisphosphate (F2,6BP) is a critical glycolytic regulator. Inhibition of the active form of PFKFB3ser461 using a novel inhibitor, PFK158 resulted in reduced glucose uptake, ATP production, lactate release as well as induction of apoptosis in gynecologic cancer cells. Moreover, we found that PFK158 synergizes with carboplatin (CBPt) and paclitaxel (PTX) in the chemoresistant cell lines, C13 and HeyA8MDR but not in their chemosensitive counterparts, OV2008 and HeyA8, respectively. We determined that PFK158-induced autophagic flux leads to lipophagy resulting in the downregulation of cPLA2, a lipid droplet (LD) associated protein. Immunofluorescence and co-immunoprecipitation revealed colocalization of p62/SQSTM1 with cPLA2 in HeyA8MDR cells uncovering a novel pathway for the breakdown of LDs promoted by PFK158. Interestingly, treating the cells with the autophagic inhibitor bafilomycin A reversed the PFK158-mediated synergy and lipophagy in chemoresistant cells. Finally, in a highly metastatic PTX-resistant in vivo ovarian mouse model, a combination of PFK158 with CBPt significantly reduced tumor weight and ascites and reduced LDs in tumor tissue as seen by immunofluorescence and transmission electron microscopy compared to untreated mice. Since the majority of cancer patients will eventually recur and develop chemoresistance, our results suggest that PFK158 in combination with standard chemotherapy may have a direct clinical role in the treatment of recurrent cancer.

Recurrence within 6 months of the last round of chemotherapy is clinically defined as platinum-resistant ovarian cancer. Gene expression associated with early recurrence may provide insights into platinum resistant recurrence. Prior studies identified a 14-gene model that accurately predicted early or late recurrence in 86% of patients. One of the genes identified was CC2D1A (encoding coiled-coil and C2 domain containing 1A), which showed higher expression in tumors from patients with early recurrence. Here, we show that CC2D1A protein expression was higher in cisplatin-resistant ovarian cancer cell lines compared to cisplatin-sensitive cell lines. In addition, immunohistochemical analysis of patient tumors on a tissue microarray (n = 146) showed that high levels of CC2D1A were associated with a significantly worse overall and progression-free survival (p = 0.0002 and p = 0.006, respectively). To understand the contribution of CC2D1A in chemoresistance, we generated shRNA-mediated knockdown of CC2D1A in SKOV3ip and PEO4 cell lines. Cell death and clonogenic assays of these isogenic clonal lines clearly showed that downregulation of CC2D1A resulted in increased sensitivity to cisplatin and paclitaxel in ovarian cancer cells. Moreover, nude mice bearing SKOV3ip xenografts with stably downregulated CC2D1A were more sensitive to chemotherapy as evidenced by a significantly longer survival time compared to xenografts derived from cells stably transduced with non-targeting shRNA. These results suggest CC2D1A promotes chemotherapy resistance in ovarian cancer.

We have previously shown that the anti-malarial compound Quinacrine (QC) inhibits ovarian cancer (OC) growth by modulating autophagy. In the present study we extended these studies to identify the molecular pathways regulated by QC to promote apoptosis independent of p53 status in OC. QC exhibited strong anti-cancer properties in OC cell lines in contrast to other anti-malarial autophagy inhibiting drugs. QC treatment selectively upregulated cell cycle inhibitor p21, and downregulated F box protein Skp2 and p62/SQSTM1 expression independent of p53 status. Genetic downregulation of key autophagy protein ATG5 abolished QC-mediated effects on both cell cycle protein p21/Skp2 as well as autophagic cargo protein p62. Furthermore, genetic silencing of p62/SQSTM1 resulted in increased sensitivity to QC-mediated apoptosis, downregulated Skp2 mRNA and increased accumulation of p21 expression. Likewise, genetic knockdown of Skp2 resulted in the upregulation of p21 and p27 and increased sensitivity of OC cells to QC treatment. In contrast, transient overexpression of exogenous p62-HA plasmid rescued the QC-mediated Skp2 downregulation indicating the positive regulation of Skp2 by p62. Collectively, these data indicate that QC-mediated effects on cell cycle proteins p21/Skp2is autophagy-dependent and p53-independent in high grade serious OC cells.

A promising new strategy for cancer therapy is to target the autophagic pathway. In the current study, we demonstrate that the antimalarial drug Quinacrine (QC) reduces cell viability and promotes chemotherapy-induced cell death in an autophagy-dependent manner more extensively in chemoresistant cells compared to their isogenic chemosensitive control cells as quantified by the Chou-Talalay methodology. Our preliminary data, in vitro and in vivo, indicate that QC induces autophagy by downregulating p62/SQSTM1 to sensitize chemoresistant cells to autophagic- and caspase-mediated cell death in a p53-independent manner. QC promotes autophagosome accumulation and enhances autophagic flux by clearance of p62 in chemoresistant ovarain cancer (OvCa) cell lines to a greater extent compared to their chemosensitive controls. Notably, p62 levels were elevated in chemoresistant OvCa cell lines and knockdown of p62 in these cells resulted in a greater response to QC treatment. Bafilomycin A, an autophagy inhibitor, restored p62 levels and reversed QC-mediated cell death and thus chemosensitization. Importantly, our in vivo data shows that QC alone and in combination with carboplatin suppresses tumor growth and ascites in the highly chemoresistant HeyA8MDR OvCa model compared to carboplatin treatment alone. Collectively, our preclinical data suggest that QC in combination with carboplatin can be an effective treatment for patients with chemoresistant OvCa.

Objective Generate preclinical data on the effect of quinacrine (QC) in inhibiting tumorigenesis in endometrial cancer (EC) in vitro and explore its role as an adjunct to standard chemotherapy in an EC mouse model. Methods Five different EC cell lines (Ishikawa, Hec-1B, KLE, ARK-2, and SPEC-2) representing different histologies, grades of EC, sensitivity to cisplatin and p53 status were used for the in vitro studies. MTT and colony formation assays were used to examine QC's ability to inhibit cell viability in vitro . The Chou-Talalay methodology was used to examine synergism between QC and cisplatin, carboplatin or paclitaxel. A cisplatin-resistant EC subcutaneous mouse model (Hec-1B) was used to examine QC's role as maintenance therapy. Results QC exhibited strong synergism in vitro when combined with cisplatin, carboplatin or paclitaxel with the highest level of synergism in the most chemo-resistant cell line. Neither QC monotherapy nor carboplatin/paclitaxel significantly delayed tumor growth in xenografts. Combination treatment (QC plus carboplatin/paclitaxel) significantly augmented the antiproliferative ability of these agents and was associated with a 14-week survival prolongation compared to carboplatin/paclitaxel. Maintenance with QC resulted in further delay in tumor progression and survival prolongation compared to carboplatin/paclitaxel. QC was not associated with weight loss and the yellow skin discoloration noted during treatment was reversible upon discontinuation. Conclusions QC exhibited significant antitumor activity against EC in vitro and was successful as maintenance therapy in chemo-resistant EC mouse xenografts. This preclinical data suggest that QC may be an important adjunct to standard chemotherapy for patients with chemo-resistant EC.

Defective autophagy and deranged metabolic pathways are common in cancer; pharmacologic targeting of these two pathways could provide a viable therapeutic option. However, how these pathways are regulated by limited availability of growth factors is still unknown. Our study shows that HSulf-1 (endosulfatase), a known tumor suppressor which attenuates heparin sulfate binding growth factor signaling, also regulates interplay between autophagy and lipogenesis. Silencing of HSulf-1 in OV202 and TOV2223 cells (ovarian cancer cell lines) resulted in increased lipid droplets (LDs), reduced autophagic vacuoles (AVs) and less LC3B puncta. In contrast, HSulf-1 proficient cells exhibit more AVs and reduced LDs. Increased LDs in HSulf-1 depleted cells was associated with increased ERK mediated cPLA2S505 phosphorylation. Conversely, HSulf-1 expression in SKOV3 cells reduced the number of LDs and increased the number of AVs compared to vector controls. Furthermore, pharmacological (AACOCF3) and ShRNA mediated downregulation of cPLA2 resulted in reduced LDs, and increased autophagy. Finally, in vivo experiment using OV202 Sh1 derived xenograft show that AACOCF3 treatment effectively attenuated tumor growth and LD biogenesis. Collectively, these results show a reciprocal regulation of autophagy and lipid biogenesis by HSulf-1 in ovarian cancer.

Heregulin (HRG), a combinatorial ligand for the epidermal growth factor receptor family, is expressed in about 30% of breast cancer tumors. HRG induces tumorigenicity and metastasis of breast cancer cells and promotes hormone-independent growth. Although HRG has been studied mostly in the context of the HRG receptor family, accumulating evidence suggests that HRG plays distinctive and causative roles in breast cancer tumorigenesis independent from the HRG receptors, demanding a comprehensive and independent study of HRG as a unique growth factor. This review provides a consolidated view of HRG and its biological role in the development, progression, and maintenance of breast cancer. Further, it provides further evidence that HRG is implicated in breast cancer resistance and targeting HRG may possibly be a beneficial tool to target a subgroup of breast carcinomas.

The expression of human Sulfatase1 (HSulf-1) is downregulated in the majority of primary ovarian cancer tumors, but the functional consequence of this downregulation remains unclear. Using two different shRNAs (Sh1 and Sh2), HSulf-1 expression was stably downregulated in ovarian cancer OV202 cells. We found that HSulf-1-deficient OV202 Sh1 and Sh2 cells formed colonies in soft agar. In contrast, nontargeting control (NTC) shRNA-transduced OV202 cells did not form any colonies. Moreover, subcutaneous injection of OV202 HSulf-1-deficient cells resulted in tumor formation in nude mice, whereas OV202 NTC cells did not. Also, ectopic expression of HSulf-1 in ovarian cancer SKOV3 cells significantly suppressed tumor growth in nude mice. Here, we show that HSulf-1-deficient OV202 cells have markedly decreased expression of proapoptotic Bim protein, which can be rescued by restoring HSulf-1 expression in OV202 Sh1 cells. Enhanced expression of HSulf-1 in HSulf-1-deficient SKOV3 cells resulted in increased Bim expression. Decreased Bim levels after loss of HSulf-1 were due to increased p-ERK, because inhibition of ERK activity with PD98059 resulted in increased Bim expression. However, treatment with a PI3 kinase/AKT inhibitor, LY294002, failed to show any change in Bim protein level. Importantly, rescuing Bim expression in HSulf-1 knockdown cells significantly retarded tumor growth in nude mice. Collectively, these results suggest that loss of HSulf-1 expression promotes tumorigenicity in ovarian cancer through regulating Bim expression.

Sulfatase 2 (Sulf-2) has been previously shown to be upregulated in breast cancer. Sulf-2 removes sulfate moieties on heparan sulfate proteoglycans which in turn modulate heparin binding growth factor signaling. Here we report that matrix detachment resulted in decreased Sulf-2 expression in breast cancer cells and increased cleavage of poly ADP-ribose polymerase. Silencing of Sulf-2 promotes matrix detachment induced cell death in MCF10DCIS cells. In an attempt to identify Sulf-2 specific inhibitor, we found that proteasomal inhibitors such as MG132, Lactacystin and Bortezomib treatment abolished Sulf-2 expression in multiple breast cancer cell lines. Additionally, we show that Bortezomib treatment of MCF10DCIS cell xenografts in mouse mammary fat pads significantly reduced tumor size, caused massive apoptosis and more importantly reduced Sulf-2 levels in vivo. Finally, our immunohistochemistry analysis of Sulf-2 expression in cohort of patient derived breast tumors indicates that Sulf-2 is significantly upregulated in autologous metastatic lesions compared to primary tumors (p < 0.037, Pearson correlation, Chi-Square analysis). In all, our data suggest that Sulf-2 might play an important role in breast cancer progression from ductal carcinoma in situ into an invasive ductal carcinoma potentially by resisting cell death. © 2013 Springer Science+Business Media Dordrecht.

Altres ajuts: We are greatly indebted to Prof. Robert A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA, USA) for providing the HMLERshCntrol and HMLERshEcad cells used in this work. Plan Nacional de I+D+I, Spain and the Departament d'Economia I Coneixement, Catalonia, Spain to Javier A. Menendez. Elisabet Cuyàs is the recipient of a "Sara Borrell" post-doctoral contract (CD15/00033, Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria -FIS-, Spain). The correction of specific signaling defects can reverse the oncogenic phenotype of tumor cells by acting in a dominant manner over the cancer genome. Unfortunately, there have been very few successful attempts at identifying the primary cues that could redirect malignant tissues to a normal phenotype. Here we show that suppression of the lipogenic enzyme fatty acid synthase (FASN) leads to stable reversion of the malignant phenotype and normalizes differentiation in a model of breast cancer (BC) progression. FASN knockdown dramatically reduced tumorigenicity of BC cells and restored tissue architecture, which was reminiscent of normal ductal-like structures in the mammary gland. Loss of FASN signaling was sufficient to direct tumors to a reversed phenotype that was near normal when considering the development of polarized growth-arrested acinar-like structure similar to those formed by nonmalignant breast cells in a 3D reconstituted basement membrane in vitro. This process, in vivo, resulted in a low proliferation index, mesenchymal-epithelial transition, and shut-off of the angiogenic switch in FASN-depleted BC cells orthotopically implanted into mammary fat pads. The role of FASN as a negative regulator of correct breast tissue architecture and terminal epithelial cell differentiation was dominant over the malignant phenotype of tumor cells possessing multiple cancer-driving genetic lesions as it remained stable during the course of serial in vivo passage of orthotopic tumor-derived cells. Transient knockdown of FASN suppressed hallmark structural and cytosolic/secretive proteins (vimentin, N-cadherin, fibronectin) in a model of EMT-induced cancer stem cells (CSC). Indirect pharmacological inhibition of FASN promoted a phenotypic switch from basal- to luminal-like tumorsphere architectures with reduced intrasphere heterogeneity. The fact that sole correction of exacerbated lipogenesis can stably reprogram cancer cells back to normal-like tissue architectures might open a new avenue to chronically restrain BC progression by using FASN-based differentiation therapies.

Inactivation of von Hippel-Lindau (VHL), a tumor suppressor gene is often associated with clear cell renal cell carcinoma (ccRCC). VHL inactivation leads to multitude of responses including enhanced growth factor signaling such as bFGF2, SDF-1α, and HGF. Here, we have identified a novel VHL-inducible gene, heparan sulfatase 2 (HSulf-2) that attenuates heparan-binding growth factor such as bFGF2 signaling. VHL-mediated HIF-1 alpha degradation was essential to restore HSulf-2 expression. Mechanistically, HSulf-2 negatively regulated vimentin expression and knockdown of vimentin abolished cell migration. This study reveals a novel layer of regulation of heparan-binding growth factor signaling via modulation of heparan sulfate by HSulf-2 in ccRCC.

HtrA1, a member of serine protease family, has been previously found to be involved in resistance to chemotherapy in ovarian cancer although the underlying mechanism is not clear. Using mixture-based oriented peptide library approach, we previously identified X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis proteins (IAPs) family as a potential substrate of HtrA1. The aim of this work is to investigate the link between HtrA1 and XIAP proteins and their relationships with chemoresistance in ovarian cancer. Our results showed that recombinant XIAP was degraded by purified wild type HtrA1 but not mutant HtrA1 in vitro. Consistent with the in vitro data, co-immunoprecipitation assays showed that HtrA1 and XIAP formed a protein complex in vivo. Ectopic expression of HtrA1 led to decreased level of XIAP in OV167 and OV202 ovarian cancer cells, while knockdown of HtrA1 resulted in increased level of XIAP in SKOV3 ovarian cancer cells. Furthermore, over-expression of HtrA1 in OV202 cells promoted cell sensitivity to cisplatin-induced apoptosis which could be reversed by increased expression of XIAP. The cleavage of XIAP induced by HtrA1 was enhanced by cisplatin treatment. Taken together, our experiments have identified XIAP as a novel substrate of HtrA1 and the degradation of XIAP by HtrA1 contributes to cell response to chemotherapy, suggesting that restoring the expression of HtrA1 may be a promising treatment strategy for ovarian cancer.

Warburg effect has emerged as a potential hallmark of many cancers. However, the molecular mechanisms that led to this metabolic state of aerobic glycolysis, particularly in ovarian cancer (OVCA) have not been completely elucidated. HSulf-1 predominantly functions by limiting the bioavailability of heparan binding growth factors and hence their downstream signaling. Here we report that HSulf-1, a known putative tumor suppressor, is a negative regulator of glycolysis. Silencing of HSulf-1 expression in OV202 cell line increased glucose uptake and lactate production by upregulating glycolytic genes such as Glut1, HKII, LDHA, as well as metabolites. Conversely, HSulf-1 overexpression in TOV21G cells resulted in the down regulation of glycolytic enzymes and reduced glycolytic phenotype, supporting the role of HSulf-1 loss in enhanced aerobic glycolysis. HSulf-1 deficiency mediated glycolytic enhancement also resulted in increased inhibitory phosphorylation of pyruvate dehydrogenase (PDH) thus blocking the entry of glucose flux into TCA cycle. Consistent with this, metabolomic and isotope tracer analysis showed reduced glucose flux into TCA cycle. Moreover, HSulf-1 loss is associated with lower oxygen consumption rate (OCR) and impaired mitochondrial function. Mechanistically, lack of HSulf-1 promotes c-Myc induction through HB-EGF-mediated p-ERK activation. Pharmacological inhibition of c-Myc reduced HB-EGF induced glycolytic enzymes implicating a major role of c-Myc in loss of HSulf-1 mediated altered glycolytic pathway in OVCA. Similarly, PG545 treatment, an agent that binds to heparan binding growth factors and sequesters growth factors away from their ligand also blocked HB-EGF signaling and reduced glucose uptake in vivo in HSulf-1 deficient cells.

The RING finger ubiquitin ligase Siah2 controls the stability of various substrates involved in stress and hypoxia responses, including the PHD3, which controls the stability of HIF-1alpha. In the present study we determined the role of Siah2 phosphorylation in the regulation of its activity toward PHD3. We show that Siah2 is subject to phosphorylation by p38 MAPK, which increases Siah2-mediated degradation of PHD3. Consistent with these findings, MKK3/MKK6 double-deficient cells, which cannot activate p38 kinases, exhibit impaired Siah2-dependent degradation of PHD3. Phosphopeptide mapping identified T24 and S29 as the primary phospho-acceptor sites. Phospho-mutant forms of Siah2 (S29A or T24A/S29A) exhibit impaired degradation of PHD3, particularly after hypoxia. Conversely, a phospho-mimic form of Siah2 (T24E/S29D) exhibits stronger degradation of PHD3, compared with wild type Siah2. Whereas phospho-mutant Siah2 exhibits weaker association with PHD3, phospho-mimic Siah2 associates as well as wild type and is localized within the perinuclear region, suggesting that phosphorylation of Siah2 affects its subcellular localization and, consequently, the degree of its association with PHD3. In all, our findings reveal the phosphorylation of Siah2 by p38 and the implications of such phosphorylation for Siah2 activity toward PHD3.

Author details Department of Experimental Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. Division of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN 55905, USA. Henry Ford Health System, Detroit, MI 48202, USA. Cancer Preventive Material Development Research Center (CPMRC), College of Oriental Medicine, Kyunghee University, Seoul 130-701, Republic of Korea. Department of Anesthesiology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. Department of Cardiovascular Disease, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. Department of Obstetrics and Gynecology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. Metabolon, Inc, Durham, NC 27713, USA. Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KN 66160, USA.

Skeletal muscle differentiation is marked by enhanced myotube formation and increased cytoskeletal rearrangement. Actin, a cytoskeletal protein is involved in various cellular functions such as glucose transport, intracellular trafficking, cell shape, and coordinated cell movement in response to various extracellular signals. The present study reveals an association between actin and p38 MAPK only in differentiated myotubes, not in proliferating myoblasts. Actin filament disassembly caused by cytochalasinD can be reversed using the potent activator of p38 MAPK, anisomycin. Pretreatment of myotubes with anisomycin partially resisted the effect of cytochalasinD. However, inhibition of p38 MAPK completely abolished the anisomycin-mediated actin remodeling. Data suggests that p38 MAPK interacts with actin and modulates actin filament rearrangement in differentiated L6E9 skeletal muscle cells.

In L6E9 skeletal muscle cells ternary complex factor (TCF) Elk-1 expression increased with the onset of skeletal muscle differentiation, whereas its activation decreased as a function of differentiation. Its expression was predominantly restricted to cytoplasm and activated ones were predominantly restricted to the nucleus of the differentiated cells. Inhibition of ERK-1/-2 activities by PD098059 resulted into significant reduction in Elk-1 expression and phosphorylation during differentiation. In contrast, inhibition of p38 mitogen-activated protein kinase (MAPK) enhanced Elk-1 expression and activation, thereby mediating inhibition of skeletal muscle differentiation. Overexpression of inactive mutant Elk-1 enhanced differentiation. Data suggest that ERK-1/-2 and p38 MAPK activities modulate Elk-1 expression and activation to regulate skeletal muscle differentiation.

Role of mitogen activated protein kinases (MAPK) in skeletal muscle differentiation is not fully understood. We investigated subtype-specific functions and their interactions, if any, in the regulation of myogenic differentiation in L6E9 skeletal muscle cells. We show inhibition of extracellular signal-regulated kinase-1 and -2 (ERK-1/-2) and activation of p38 MAP kinase during the differentiation of L6E9 rat skeletal muscle cells under low serum condition. Inhibition of ERK-1/-2 activity dramatically enhanced differentiation as was evident from cellular morphology, expression of muscle differentiation specific marker proteins, suggesting that ERK-1/-2 activation may be inhibitory to initiation and progression of differentiation. In contrast, inhibition of p38 MAP kinase completely prevented differentiation; meaning p38 activation is required from the initiation till terminal differentiation of L6E9 cells. Moreover, inhibition of ERK-1/-2 activities enhanced the activation of p38 MAP kinase that resulted in enhancement of differentiation; whereas inhibition of p38 MAP kinase activity enhanced the ERK-1/-2 activities culminating in abrogation of differentiation. We conclude that ERK-1/-2 and p38 MAP kinase cascades oppositely regulate each other's function(s) thereby regulating L6E9 skeletal muscle differentiation.

Heparan sulfate proteoglycans (HSPGs) are an integral and dynamic part of normal tissue architecture at the cell surface and within the extracellular matrix (ECM). The modification of HSPGs in the tumour microenvironment is known to result not just in structural but also functional consequences which significantly impact cancer progression. As substrates for the key enzymes sulfatases and heparanase, the modification of HSPGs is typically characterised by the degradation of heparan sulfate (HS) chains/sulfation patterns via the endo–6-O-sulfatases (Sulf1 and Sulf2) or by heparanase, an endo-glycosidase that cleaves the HS polymers releasing smaller fragments from HSPG complexes. Numerous studies have demonstrated how these enzymes actively influence cancer cell proliferation, signalling, invasion and metastasis. The activity or expression of these enzymes have been reported to be modified in a variety of cancers. Such observations are consistent with the degradation of normal architecture and basement membranes which are typically compromised in metastatic disease. Moreover, recent studies elucidating the requirements for these proteins in tumour initiation and progression exemplify their importance in the development and progression of cancer. Thus, as the influence of the tumour microenvironment in cancer progression becomes more apparent, the focus on targeting enzymes that degrade HSPGs highlights one approach to maintain normal tissue architecture, inhibit tumour progression and block metastasis. This review discusses the role of these enzymes in the context of the tumour microenvironment and their promise as therapeutic targets for the treatment of cancer.

Background Loss of the endosulfatase HSulf-1 is common in ovarian cancer, upregulates heparin binding growth factor signaling and potentiates tumorigenesis and angiogenesis. However, metabolic differences between isogenic cells with and without HSulf-1 have not been characterized upon HSulf-1 suppression in vitro. Since growth factor signaling is closely tied to metabolic alterations, we determined the extent to which HSulf-1 loss affects cancer cell metabolism. Results Ingenuity pathway analysis of gene expression in HSulf-1 shRNA-silenced cells (Sh1 and Sh2 cells) compared to non-targeted control shRNA cells (NTC cells) and subsequent Kyoto Encyclopedia of Genes and Genomics (KEGG) database analysis showed altered metabolic pathways with changes in the lipid metabolism as one of the major pathways altered inSh1 and 2 cells. Untargeted global metabolomic profiling in these isogenic cell lines identified approximately 338 metabolites using GC/MS and LC/MS/MS platforms. Knockdown of HSulf-1 in OV202 cells induced significant changes in 156 metabolites associated with several metabolic pathways including amino acid, lipids, and nucleotides. Loss of HSulf-1 promoted overall fatty acid synthesis leading to enhance the metabolite levels of long chain, branched, and essential fatty acids along with sphingolipids. Furthermore, HSulf-1 loss induced the expression of lipogenic genes including FASN, SREBF1, PPARγ, and PLA2G3 stimulated lipid droplet accumulation. Conversely, re-expression of HSulf-1 in Sh1 cells reduced the lipid droplet formation. Additionally, HSulf-1 also enhanced CPT1A and fatty acid oxidation and augmented the protein expression of key lipolytic enzymes such as MAGL, DAGLA, HSL, and ASCL1. Overall, these findings suggest that loss of HSulf-1 by concomitantly enhancing fatty acid synthesis and oxidation confers a lipogenic phenotype leading to the metabolic alterations associated with the progression of ovarian cancer. Conclusions Taken together, these findings demonstrate that loss of HSulf-1 potentially contributes to the metabolic alterations associated with the progression of ovarian pathogenesis, specifically impacting the lipogenic phenotype of ovarian cancer cells that can be therapeutically targeted.

The expression of human Sulfatase1 (HSulf-1) is downregulated in the majority of primary ovarian cancer tumors, but the functional consequence of this downregulation remains unclear. Using two different shRNAs (Sh1 and Sh2), HSulf-1 expression was stably downregulated in ovarian cancer OV202 cells. We found that HSulf-1-deficient OV202 Sh1 and Sh2 cells formed colonies in soft agar. In contrast, nontargeting control (NTC) shRNA-transduced OV202 cells did not form any colonies. Moreover, subcutaneous injection of OV202 HSulf-1-deficient cells resulted in tumor formation in nude mice, whereas OV202 NTC cells did not. Also, ectopic expression of HSulf-1 in ovarian cancer SKOV3 cells significantly suppressed tumor growth in nude mice. Here, we show that HSulf-1-deficient OV202 cells have markedly decreased expression of proapoptotic Bim protein, which can be rescued by restoring HSulf-1 expression in OV202 Sh1 cells. Enhanced expression of HSulf-1 in HSulf-1-deficient SKOV3 cells resulted in increased Bim expression. Decreased Bim levels after loss of HSulf-1 were due to increased p-ERK, because inhibition of ERK activity with PD98059 resulted in increased Bim expression. However, treatment with a PI3 kinase/ AKT inhibitor, LY294002, failed to show any change in Bim protein level. Importantly, rescuing Bim expression in HSulf-1 knockdown cells significantly retarded tumor growth in nude mice. Collectively, these results suggest that loss of HSulf-1 expression promotes tumorigenicity in ovarian cancer through regulating Bim expression.

Although the majority of patients with endometrial cancer (EC) are diagnosed early when disease is confined in the uterus and prognosis is excellent, there is a subset of patients with advanced, metastatic, recurrent or persistent after surgery disease with dismal prognosis. Chemotherapy has a critical role for these patients. Carboplatin (carbo) and paclitaxel (taxol) is the standard first-line chemotherapy in EC with response rates as low as 50% and even lower for recurrent disease. Furthermore, many patients will eventually relapse with chemo-resistant disease and very poor prognosis. As the chemotherapeutic options for these patients are limited and of questionable efficacy, it is of paramount importance that novel agents are identified to increase or restore chemo-sensitization to platinum-based chemotherapy. Quinacrine (QC) is an inexpensive antimalarial drug with a predictable safety profile which recently surfaced as a promising anticancer agent thought to be associated with decreased risk of developing chemo-resistance through targeting multiple pathways simultaneously. While prior investigators have provided evidence that QC exerts strong anticancer activity against a wide range of solid tumors, there are no studies focused on QC in EC. In our study, QC exhibited strong synergism in vitro when combined with cisplatin, carbo or taxol with the highest level of synergism being observed in the most chemo-resistant EC cell line. Neither QC monotherapy (QC every other day for 3 weeks) nor standard chemotherapy (carbo+taxol for 3 cycles, on days 3, 7, 11) significantly delayed tumor growth in the mouse xenografts. Only combination (standard chemo + QC every other day during carbo+taxol treatment) and maintenance (combination regimen followed by QC every other day until end of study) significantly augmented carbo+taxol antiproliferative effect as evidenced by the significant decrease in tumor burden and delayed tumor growth in xenografts. Combination treatment was associated with a 14-day prolongation of median survival compared to carbo+taxol (68 vs. 54 days). Maintenance therapy with QC was proven superior to carbo+taxol as it resulted in long-term stabilization of disease and further prolongation of overall survival; in fact, median survival in the maintenance group was not reached as the mice were surviving at the end of study. QC alone, in combination with carbo+taxol or as maintenance was well-tolerated with no issues of weight loss compared to control mice. A yellow skin discoloration was noted during QC treatment which was entirely reversible within few days upon QC discontinuation. In conclusion, QC exhibited significant antitumor activity against EC cell lines in vitro and was successful in stabilizing disease and prolonging survival as maintenance therapy in chemo-resistant EC mouse xenografts. This data suggests that QC may be an important adjunct to standard chemotherapy for patients with recurrent EC. Citation Format: Eleftheria Kalogera, Debarshi Roy, Ashwani Khurana, Susmita Mondal, Xiaoping He, Sean C. Dowdy, Viji Shridhar. Quinacrine in endometrial cancer: repurposing an old antimalarial drug. [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 261.

Resistance to chemotherapy is a major hurdle that results in the increased mortality associated with ovarian cancer (OvCa). New therapeutics that synergize with cis-carboplatin/paclitaxel in recurrent tumors will have an enormous impact in prolonging the survival of OvCa patients. Due to the enormous cost of designing and testing new drugs, we have adopted the drug repurposing approach and tested the effect of the antimalarial drug quinacrine (QC) in sensitizing drug resistant OvCa cells to carboplatin induced cytotoxicity and determined that it is autophagy dependent. We have discovered that QC, an autophagy modulator exhibited distinct anti-cancer properties in OvCa cell lines in contrast to other anti-malarial/autophagy inhibitor drugs such as hydroxyquinacrine, bafilomycin A and 3-MA. It is well established that autophagy can be a survival mechanism under stress in cancer or a death promoting phenomenon that is cell type and contextual dependent. Here we demonstrate that QC acts to promote chemotherapy induced cell death specifically more in resistant cells in an autophagy dependent manner. Our preliminary data both in vitro and in vivo indicate that QC induced autophagy downregulates p62 and upregulates cathepsin L levels to sensitize the chemoresistant cells to autophagic and caspase mediated cell death in a p53 independent manner. These unexpected findings led us to explore the mechanistic basis of QC-induced upregulation of the cell cycle inhibitor p21 in cells bearing mutant p53. We uncovered that irrespective of p53 status, QC induces p21 expression in an autophagy-dependent p62-Skp2 mediated ubiquitin pathway resulting in cell cycle arrest and cell death. Since QC is already in PhaseI Clinical trial, these studies may spur new treatment strategies to improve OvCa patient outcome and patient care. Citation Format: Debarshi Roy, Ashwani Khurana, Eleftheria Kalogera, Xuyang Wen, Susmita Mondal, Viji Shridhar. Can we repurpose anti-malarial drug quinacrine to treat ovarian cancer? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B74.

Inactivation of von Hippel-Lindau (VHL), a tumor suppressor gene is often associated with clear cell renal cell carcinoma (ccRCC). VHL inactivation leads to multitude of responses including enhanced growth factor signaling such as bFGF2, SDF-1α, and HGF. Here, we have identified a novel VHL-inducible gene, heparan sulfatase 2 (HSulf-2) that attenuates heparan-binding growth factor such as bFGF2 signaling. VHL-mediated HIF-1 alpha degradation was essential to restore HSulf-2 expression. Mechanistically, HSulf-2 negatively regulated vimentin expression and knockdown of vimentin abolished cell migration. This study reveals a novel layer of regulation of heparan-binding growth factor signaling via modulation of heparan sulfate by HSulf-2 in ccRCC.

HSulf-1 modulates the sulfation states of heparan sulfate proteoglycans critical for heparin binding growth factor signaling. In the present study, we show that HSulf-1 is transcriptionally deregulated under hypoxia in breast cancer cell lines. Knockdown of HIF-1α rescued HSulf-1 downregulation imposed by hypoxia, both at the RNA and protein levels. Chromatin immunoprecipitation with HIF-1α and HIF-2α antibodies confirmed recruitment of HIF-α proteins to the two functional hypoxia-responsive elements on the native HSulf-1 promoter. HSulf-1 depletion in breast cancer cells resulted in an increased and sustained bFGF2 (basic fibroblast growth factor) signaling and promoted cell migration and invasion under hypoxic conditions. In addition, FGFR2 (fibroblast growth factor receptor 2) depletion in HSulf-1–silenced breast cancer cells attenuated hypoxia-mediated cell invasion. Immunohistochemical analysis of 53 invasive ductal carcinomas and their autologous metastatic lesions revealed an inverse correlation for the expression of HSulf-1 to CAIX in both the primary tumors (P ≥ 0.0198) and metastatic lesions (P ≥ 0.0067), respectively, by χ2 test. Finally, HSulf-1 expression levels in breast tumors by RNA in situ hybridization showed that high HSulf-1 expression is associated with increased disease-free and overall survival (P ≥ 0.03 and P ≥ 0.0001, respectively). Collectively, these results reveal an important link between loss of HSulf-1 under hypoxic microenvironment and increased growth factor signaling, cell migration, and invasion. Cancer Res; 71(6); 2152–61. ©2011 AACR.

Purpose: The purpose of this study was to determine if loss of serine protease HtrA1 in endometrial cancer will promote the invasive potential of EC cell lines. Experimental design: Western blot analysis and immunohistochemistry methods were used to determine HtrA1 expression in EC cell lines and primary tumors, respectively. Migration, invasion assays and in vivo xenograft experiment were performed to compare the extent of metastasis between HtrA1 expressing and HtrA1 knocked down clones. Results: Western blot analysis of HtrA1 in 13 EC cell lines revealed complete loss of HtrA1 expression in all seven papillary serous EC cell lines. Downregulation of HtrA1 in Hec1A and Hec1B cell lines resulted in a three- to fourfold increase in the invasive potential. Exogenous expression of HtrA1 in Ark1 and Ark2 cells resulted in three- to fourfold decrease in both invasive and migration potential of these cells. There was an increased rate of metastasis to the lungs associated with HtrA1 downregulation in Hec1B cells compared to control cells with endogenous HtrA1 expression. Enhanced expression of HtrA1 in Ark2 cells resulted in significantly less tumor nodules metastasizing to the lungs compared to parental or protease deficient (SA mutant) Ark2 cells. Immunohistochemical analysis showed 57% (105/184) of primary EC tumors had low HtrA1 expression. The association of low HtrA1 expression with high-grade endometrioid tumors was statistically significant (P = 0.016). Conclusions: Collectively, these data indicate loss of HtrA1 may contribute to the aggressiveness and metastatic ability of endometrial tumors. Clin Cancer Res; 17(3); 427–36. ©2010 AACR.

We recently identified HSulf-1 as a down-regulated gene in ovarian carcinomas. Our previous analysis indicated that HSulf-1 inactivation in ovarian cancers is partly mediated by loss of heterozygosity and epigenetic silencing. Here, we show that variant hepatic nuclear factor 1 (vHNF1), encoded by transcription factor 2 gene (TCF2, HNF1β), negatively regulates HSulf-1 expression in ovarian cancer. Immunoblot assay revealed that vHNF1 is highly expressed in HSulf-1–deficient OV207, SKOV3, and TOV-21G cell lines but not in HSulf-1–expressing OSE, OV167, and OV202 cells. By short hairpin RNA–mediated down-regulation of vHNF1 in TOV-21G cells and transient enhanced vHNF1 expression in OV202 cells, we showed that vHNF1 suppresses HSulf-1 expression in ovarian cancer cell lines. Reporter assay and chromatin immunoprecipitation experiments showed that vHNF1 is specifically recruited to HSulf-1 promoter at two different vHNF1-responsive elements in OV207 and TOV-21G cells. Additionally, down-regulation of vHNF1 expression in OV207 and TOV-21G cells increased cisplatin- or paclitaxel-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays and this effect was reversed by down-regulation of HSulf-1. Moreover, nude mice bearing TOV-21G cell xenografts with stably down-regulated vHNF1 were more sensitive to cisplatin- or paclitaxel-induced cytotoxicity compared with xenografts of TOV-21G clonal lines with nontargeted control short hairpin RNA. Finally, immunohistochemical analysis of 501 ovarian tumors including 140 clear-cell tumors on tissue microarrays showed that vHNF1 inversely correlates to HSulf-1 expression. Collectively, these results indicate that vHNF1 acts as a repressor of HSulf-1 expression and might be a molecular target for ovarian cancer therapy. [Cancer Res 2009;69(11):4843–50]

Metabolic alterations in cancer are increasingly being recognized as important novel targets. Among different regulators of altered metabolism, 6-PhosphoFructo-2-Kinase/Fructose-2,6-Bisphosphatases 3 (PFKFB3) a glycolytic enzyme required for tumorigenic growth, and produces fructose-2,6-bisphosphate (F2,6BP), is recognized as important regulator of glycolysis. We determined that the active form of PFKFB3ser471 was overexpressed in chemo-resistant ovarian cancer (OVCA) cell lines and resistant patient derived xenograft (PDX) models compared to their isogenic chemo-sensitive cell lines and sensitive PDX models. These results suggest that targeting PFKFB3 will have beneficial effects in patients with OVCA. PFK-158 is a potent and selective inhibitor of PFKFB3 that is currently being investigated in a Phase 1 study in patients with advanced solid malignancies. Here we report for the first time, PFK-158 dose-dependently inhibits cell proliferation and colony formation ability in both carboplatin and paclitaxel (PTX) resistant cell lines. PFK-158 markedly suppresses glycolysis by reducing glucose uptake, ATP production, lactate dehydrogenase activity and lactate secretion. Interestingly, both Chou-Talalay synergy analysis and apoptosis assay by annexin V-PI staining showed PFK-158 strongly synergies with both PTX and carboplatin in PTX and cisplatin resistant cell lines respectively. PFK-158 induced apoptosis through a significant increase in caspase-3/7 activation and PARP cleavage. Moreover, in a highly metastatic PTX-resistant in vivo model, combination of PFK-158 with carboplatin and PTX significantly reduced tumor weight, ascites and metastasis compared to untreated and PFK-158, carboplatin or PTX alone treated mice. Since the majority of OVCA patients recur with chemo-resistant disease, our results suggest that PFK-158 in combination with standard chemotherapy may have clinical utility in late stage ovarian cancer patients. Citation Format: Susmita Mondal, Debarshi Roy, Eleftheria Kalogera, Ashwani Khurana, Gilles H. Tapolsky, Sucheta Telang, Jason Chesney, Viji Shridhar. Inhibition of PFKFB3/glycolysis overcomes chemoresistance in ovarian cancers. [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 2706. doi:10.1158/1538-7445.AM2015-2706

We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associated membrane protein), a predicted seven-transmembrane protein that is localized primarily within the plasma membrane and associates with JNK1 through its C-terminal domain. JAMP association with JNK1 outcompetes JNK1 association with mitogen-activated protein kinase phosphatase 5, resulting in increased and prolonged JNK1 activity following stress. Elevated expression of JAMP following UV or tunicamycin treatment results in sustained JNK activity and a higher level of JNK-dependent apoptosis. Inhibition of JAMP expression by RNA interference reduces the degree and duration of JNK activation and concomitantly the level of stress-induced apoptosis. Through its regulation of JNK1 activity, JAMP emerges as a membrane-anchored regulator of the duration of JNK1 activity in response to diverse stress stimuli.

Previous studies on skeletal muscle differentiation showed that myogenesis is regulated by extracellular signal-regulated kinases (ERK-1/-2) and p38 mitogen activated kinase (MAPK) pathways. Present study shows that c-Jun NH(2)-terminal protein kinase (JNK) activities were up regulated during skeletal muscle differentiation in rat skeletal muscle L6E9 cells, as determined by Western immunoblot of differentiating cells probed with anti-phospho-JNK antibody. Inhibition of JNK activities by JNK inhibitor II drastically inhibited differentiation as determined by decreased myosin, myogenin expression and creatine kinase activity. The inhibition of the differentiation was regulated by apoptosis as determined by the detection of poly(ADP-ribose) polymerase (PARP) cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells when JNK activities were inhibited. Apoptosis was accompanied by marked expression and activation of c-Jun and p53 transcription factors. Taken together, our results indicate that basal JNK activities are essential for regulating skeletal muscle differentiation, and inhibition of JNK activation affects myogenesis by apoptosis dependent on c-Jun and p53 transcription factors.

Role of mitogen activated protein kinases (MAPK) in skeletal muscle differentiation is not fully understood. We investigated subtype-specific functions and their interactions, if any, in the regulation of myogenic differentiation in L6E9 skeletal muscle cells. We show inhibition of extracellular signal-regulated kinase-1 and -2 (ERK-1/-2) and activation of p38 MAP kinase during the differentiation of L6E9 rat skeletal muscle cells under low serum condition. Inhibition of ERK-1/-2 activity dramatically enhanced differentiation as was evident from cellular morphology, expression of muscle differentiation specific marker proteins, suggesting that ERK-1/-2 activation may be inhibitory to initiation and progression of differentiation. In contrast, inhibition of p38 MAP kinase completely prevented differentiation; meaning p38 activation is required from the initiation till terminal differentiation of L6E9 cells. Moreover, inhibition of ERK-1/-2 activities enhanced the activation of p38 MAP kinase that resulted in enhancement of differentiation; whereas inhibition of p38 MAP kinase activity enhanced the ERK-1/-2 activities culminating in abrogation of differentiation. We conclude that ERK-1/-2 andp38 MAPkinase cascades oppositelyregulate each other's function(s) thereby regulating L6E9 skeletal muscle differentiation.

Increased de novo lipogenesis due to enhanced expression of several lipogenic enzymes has been found in many types of human cancers. Fatty acid synthase (FASN), the key enzyme in the de novo lipogenesis pathway, is one of the most commonly over-expressed lipogenic enzymes. Studies in the past decade have recognized FASN overexpression as a molecular marker for poor prognosis and malignant phenotype of many cancers. Tumor-associated FASN, by conferring growth and survival advantages rather than functioning as an anabolic energy-storage pathway, has also been suggested to play an active part in the development, maintenance, and metastatic progression of human cancers. More recently, FASN has been implicated in contributing to cellular resistance to several anticancer treatments. A variety of agents have been developed to target lipogenic enzymes and the key regulators involved in lipid metabolism in cancer cell for therapeutic purposes. The development of several FASN inhibitors has been reported from both academic labs and industries. When used in in vitro, xenograft and genetically induced mouse model studies, these inhibitors have supported FASN as an excellent target. A new generation of FASN inhibitors was developed at 3-V Biosciences that are highly specific, reversible inhibitors of FASN. One compound in this series, TVB-2640, is orally bioavailable and has progressed into Phase 1 clinical studies. The effect of two anti-FASN drugs in this series, TVB-3150 and TVB-3199 were tested for their effect on breast cancer cells. We demonstrated that the EC50 is between 9-50 nM, depending on the drug and the tested cell line. We then demonstrated that the drugs inhibited the anchorage-dependent and -independent growth of FASN expressing cells in a dose dependent manner. The apparent mechanism by which the FASN inhibitors promote inhibition of growth is by inducing apoptotic cell death in a time- and dose-dependent manner that involves regulation of apoptotic and pro-apoptotic proteins. Furthermore, the combination of the anti-FASN drugs with small molecule inhibitors of the antiapoptotic proteins Bcl-2 and Bcl-xL significantly increased the apoptotic effect in a synergistic manner compared to either of the synthetic inhibitors alone. In vivo studies demonstrate tumor growth inhibition without toxicity. Clinical development of a first-in-class, orally active, potent, and reversible FASN inhibitor as a novel cancer therapeutic is ongoing. Citation Format: Ruth Lupu, Anatilde Gonzalez Guerrico, Ashwani Khurana, Timothy Heuer, George Kemble, Chandra Mohan KVP. Small-molecule FASN inhibitors promote growth inhibition growth and apoptosis of breast cancer. [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 2713. doi:10.1158/1538-7445.AM2014-2713

Heregulin (HRG)s are high-affinity combinatorial ligands for the HER family of tyrosine kinase receptors. HRG induces heterodimerization and cross-talk between members of the HER tyrosine kinase receptor family. HRG interaction with HER3 induces heterodimerization between HER3 and other HER receptors, including epidermal growth factor receptor (EGFR) and HER2, resulting in cross-talk-induced tyrosine phosphorylation. Several studies have revealed that HRG is expressed in about 40% of invasive breast carcinomas, many of which are triple negative breast carcinomas. We have previously demonstrated that HRG expression induces tumorigenesis and metastasis of MCF-7 cells. We tested the effect of HRG on non-committed MCF10A cells. MCF-10A cell line is an immortalized, non-transformed epithelial cell line derived from human fibrocystic mammary tissue. These cells lack the ability to form tumors in nude mice and the ability to grow in anchorage-independent assays. We found that forced expression of HRG in MCF10A induced cellular transformation, while the control MCF10A/Vector cells did not growth. MCF10A/HRG cells express constitutive phosphorylation of HER3 and induction of the signaling cascade. HER3 phosphorylation occurs as a result of HRG induction of HER3 heterodimerization with EGFR in an autocrine manner. Lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2 and EGFR pathways, blocked HER3 phosphorylation and the resulted signaling in MCF10A/HRG cells and prevented their anchorage-independent growth demonstrating that HER3 phosphorylation is responsible, at least in part, for the transformation of MCF10A/HRG cells. This study provides an alternate targeting prospective for breast carcinomas that express HER3 and HRG by targeting the autocrine-loop (HRG/HER3) and thus preventing HRG from binding to HER3, disrupting the possible receptor heterodimerization, and leading to blockage of the proliferative signals and promoting inhibition of growth. Citation Format: Ruth Lupu, Ashwani Khurana, Ghiara Lugo, Anatilde Gonzalez Guerrico. Heregulin induces transformation of mammary epithelial cells via HER3 activation. [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 3506. doi:10.1158/1538-7445.AM2014-3506

We have previously shown that a frequent loss of endosulfatase HSulf-1 in ovarian cancer enhances the heparin binding growth factor signaling which subsequently induces and potentiates tumorigenesis. Our recent data indicate that knockdown of HSulf-1 in ovarian cancer cells significantly alter levels of several metabolites which is comparable to the deranged metabolite profiles in primary ovarian tumors. Here we report that the metabolites associated with lipid metabolism are altered in such a way that the cells start manifesting a “lipogenic phenotype” characterized by an increased fatty acid synthesis, storage and mitochondrial beta oxidation. The lipogenic phenotype is associated with an accumulation of cytoplasmic lipid droplets (LDs) in a stably HSulf-1 knockdown cells [OV202 (Sh1/2) and OV2223 cells]. Presence of LDs is evidenced by BODIPY staining and transmission electron microscopy (TEM) analysis in Sh1/2 and a non-transduced control (NTC) cells. Conversely, enhanced expression of HSulf-1 in TOV21G, SKOV3, and C13 cells also resulted in reducing the number of LDs. More importantly, MEFs (mouse embryonic fibroblasts) from HSulf-1 knockout mice also showed an increased LD biogenesis compared to the wild type MEFs. Microarray analysis identified cytoplasmic phospholipase A2 (cPLA2; an enzyme involved in LD biogenesis) as one of the genes that was significantly upregulated in OV202 Sh1 and Sh2 cells. We found that Sh1 and Sh2 cells show an increased phosphorylation (ser505) of cytoplasmic phospholipase A2 (p-cPLA2) compared to NTC cells. Phosphorylation at ser505 is vital for the enzyme activity of cPLA2 and also necessary for LD biogenesis. We inhibited the LD biogenesis in Sh1 cells by a pharmacological inhibitor of cPLA2 (AACOCF3 and MAFP) as well by shRNA mediated knockdown of endogenous cPLA2. Further, we stimulated NTC and Sh1 cells with heparin binding growth factor (50ng/ml) for different time points and found an elevated phosphorylation of cPLA2 and extracellular signal related kinase (ERK), which is also inhibited by a pre-treatment of cells with cPLA2 inhibitor. Interestingly, inhibition of phospho-ERK by U0126 (MEK inhibitor) also inhibits the phosphorylation of cPLA2. These findings strongly suggest that loss of HSulf-1 promotes lipid metabolism in the ovarian cancer cells via activation of cPLA2 via an ERK dependent pathway. The present work highlights a novel role HSulf-1 in regulating lipid metabolism which could lead to a better understanding of tumorigenesis and identification of new targets for tumor therapy. Citation Format: Debarshi Roy, Xiaoping He, Ashwani Khurana, Susmita Mondal, Deok-Beom Jung, Sung Kim, Thomas Dierks, Clifford Folmes, Andre Terzic, Viji Shridhar. HSulf-1 loss in ovarian cancer cells induces lipid droplet biogenesis. [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 2439. doi:10.1158/1538-7445.AM2014-2439

In a recent issue of Cell Cycle, Menendez and colleagues proposed a novel concept, that metformin is synthetically lethal with glucose withdrawal in cancer cells.1 Historically, synthetic lethality has focused on how tumor cells are responsive to certain agents that only harbor specific constitutive epigenetic or genetic lesions.2 More recent data from several groups have uncovered that altered tumor microenvironment could be used to confer synthetic lethality to specific drugs, defined as “contextual synthetic lethality,” that is microenvironment-mediated. For example, hypoxia-induced HR (homologous repair) defect has been shown to be synthetically lethal to PARP inhibition, while PARP inhibition, per se, did not alter HR inhibition or function, thus providing a prime example of “contextual synthetic lethality.”3 In this report, Menendez et al. have elegantly connected the glucose-deprived tumor microenvironment in primary tumors as a synthetic lethal partner to metformin. Metformin is a FDA-approved drug to treat diabetic patients that is gaining momentum as a repurposing drug for cancer treatment.4 Using several different breast cancer cells with and without oncogenic activation, the authors have shown that the glucose-rich conditions of the in vitro experiments dictates the use of very high concentrations of metformin, which are not applicable to glucose-starved in vivo conditions. While other reports have alluded to the effect of glucose withdrawal in killing genetically compromised cells to therapeutic effect of metformin in vitro,5 Menendez et al have provided a logical explanation for the use of very high concentrations of metformin to achieve anticancer effects in vitro in the high glucose-rich environment used in these experiments, which are clinically not applicable in vivo in patients. Based on these findings, it can be envisaged that in the tumor microenvironment, where the cancer cells are under extreme nutritional and hypoxic stress (a niche for cancer stem cells), metformin treatment could favor synthetic lethality and hence effectively can attenuate tumor growth. The tumor microenvironment thus enables the bioenergetic switch in favor of glycolysis and dependence on glucose and glutamine as a rapid source of nutrition. While the authors’ data clearly depicts how metformin eliminates the tolerance of the breast cancer cells to fluctuations in glucose concentrations, it is important to understand how the availability of other dominant sources of energy, such as glutamine, might participate in this scenario. It is plausible that subtype of breast cancers, i.e., basal vs luminal, might depend on different energy sources, albeit to a different extent.6 This is important, because tumor cells often acquire metabolic adaptability toward available preferred energy source to adapt well to nutritional stress via autophagy and altered metabolism.7 Along these lines, the authors rationalize the therapeutic targeting of the cancer stem cells by metformin through its synthetic lethal activity to the hyperglycotic phenotype often seen in CSC to sustain their stemness.8 Further characterization of how metformin treatment alters the metabolic nodes in cancer stem cells and/or p53-null cells would explain the underpinning mechanisms for increased susceptibility of these indolent and aggressive cancer cells toward metformin. It is well documented that metformin, by inhibiting complex I of respiratory chain in mitochondria (ETCI), induces a decrease in the ATP levels, and that glucose depletion also decreases ATP levels, albeit to varying levels. Therefore, it is possible that simultaneous targeting of both pathways (glycolytic pathway and OXPHOS) caused ATP depletion below a critical threshold, resulting in cell death. This concept is supported by the elegant study9 highlighting the effectiveness of combination of glycolysis inhibition by 2-DG and metformin in several preclinical models exhibiting anti-tumor effects, including MB-MDA231 used in this study. Since recent studies indicate that inhibiting glucose uptake with small-molecule inhibitors led to a decline in cylcin E2 and p-RB levels,10 it is a possibility that cell cycle inhibitor levels are also regulated under glucose withdrawal conditions, sensitizing cells to cytotoxic effects of metformin in breast cancer cells. Considering data from several studies, a view that metformin treatment has pleotropic effects on several signaling pathways under glucose-free conditions seems a practical possibility. Overall, this work offers several new insights into glucose-dependent mechanisms underpinning the mode of action of metformin as a viable therapeutic strategy.

Introduction Ductal carcinoma in situ (DCIS) of the breast is a heterogeneous group of proliferative cellular lesions that have the potential to become invasive. Very little is known about the molecular alterations involved in the progression from DCIS to invasive ductal carcinoma (IDC). Heparan endosulfatase (HSulf-2) edits sulfate moieties on heparan sulfate proteoglycans (HSPGs) and has been implicated in modulating heparin binding growth factor signaling, angiogenesis and tumorigenesis. However, the role of HSulf-2 in breast cancer progression is poorly understood. MCF10DCIS.com cells (referred as MCF10DCIS) express HSulf-2 and form comedo type DCIS and progress to IDC when transplanted in immune-deficient mice and, therefore, is an ideal model to study breast cancer progression. We evaluated the role of HSulf-2 in progression from DCIS to IDC using mouse fat pad mammary xenografts. Methods Non-target control (NTC) and HSulf-2 knockdown in MCF10DCIS breast cancer cells were achieved by NTC shRNA and two different lentiviral shRNA against HSulf-2 respectively. Xenografts were established by injecting NTC and HSulf-2 deficient MCF10DCIS cells in mouse mammary fat pads. Xenografts were subjected to H&E staining for morphological analysis, TUNEL and Propidium iodide staining (to determine the extent of apoptosis), Western blot analysis and zymography. Results Using a mouse mammary fat pad derived xenograft model, we observed that compared to control treated xenografts, down-regulation of HSulf-2 was associated with significant delays in growth at Week 7 (P-value < 0.05). Histological examination of the tumors demonstrated substantial differences in comedo necrosis, with marked luminal apoptosis and up-regulation of apoptotic markers Bim, cleaved PARP and cleaved caspase 3 in HSulf-2 depleted xenografts. Furthermore, HSulf-2 depleted xenografts retained the basement membrane integrity with decreased activity and expression of matrix metalloproteinase 9 (MMP-9), an enzyme critical for degradation of extracellular matrix compared to nontargeted control. Conclusion Our data suggest that HSulf-2 expression may be critical for human breast cancer progression. Down-regulation of HSulf-2 leads to retention of comedo type DCIS and delays the progression of DCIS to IDC. Further studies are necessary to determine if therapeutic targeting of HSulf-2 expression might delay the progression of DCIS to IDC.

We previously identified serine protease HtrA1 as a down-regulated gene in epithelial ovarian cancer (EOC) that modulates cisplatin-induced cytotoxicity. However, the mechanism by which HtrA1 sensitizes cells to cisplatin-induced cytotoxicity remained unknown. We have shown previously that the pan-cyclin dependent kinase inhibitor flavopiridol (FP) sensitizes cells to cisplatin-induced cytotoxicity; we hypothesized that flavopiridol might therefore modulate the expression of pro-apoptotic proteins. Microarray data indicated that while flavopiridol treatment alone or in combination with cisplatin up-regulated HtrA1 expression by 2-3 fold (providing a means to upregulate HtrA1 expression), treatment with cisplatin alone had no effect. Interestingly, in searching for underlying mechanisms, we noted that WT-1 transcription factor binding elements that is present in HtrA1 promoter is down-regulated by flavopiridol in our microarray studies. Immunoblot analysis revealed that WT-1 is highly expressed in HtrA1-deficient OV167, OV202, PE04 and Ovcar3 cells but not in HtrA1-expressing SKOV3, TOV21G and PE01 cells, suggesting WT-1 negatively regulates HtrA1 expression in EOC. Immunoblot analysis showed FP treatment resulted in down-regulation of WT-1 with a corresponding increase in HtrA1 protein levels in OV202 cells. Additionally, stable down-regulation of WT-1 by short hairpin RNA (shRNA) in OV202 cells increased HtrA1 expression. Conversely, enhanced expression of full length WT-1 isoform in SKOV3 cells downregulated HtrA1 expression. Reporter assay showed that FP treatment upregulated HtrA1 promoter activity in OV202 cells. Reporter assay and chromatin immunoprecipitation experiments showed that WT-1 is specifically recruited to the HtrA1 promoter. Moreover, downregulation of WT-1 expression in OV202 cells increased cisplatin-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays – an effect reversed by enhanced expression of HtrA1. Collectively, our studies demonstrate a linkage between the effects of flavopiridol on HtrA1 and its regulatory transcription factor WT-1 that appears in part to explain why flavopiridol has chemosensitizing properties 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 4015. doi:10.1158/1538-7445.AM2011-4015

We have previously identified serine protease HtrA1 as a down-regulated gene in ovarian cancer and loss of HtrA1 may contribute to cell resistance to cisplatin treatment. However, the exact mechanism by which HtrA1 regulates chemosensitivity in ovarian cancer is currently unknown. Here, we used mixture-based oriented peptide library approach and identified XIAP, an important member of the inhibitor of apoptosis (IAP) protein family, as one possible substrate of HtrA1. Recombinant wild type (WT) HtrA1 but not protease-inactive SA mutant HtrA1 can cleave XIAP protein in vitro. Over-expression of HtrA1 led to decreased level of XIAP in OV167 and OV202 ovarian cancer cells, while down-regulation of HtrA1 resulted in increased level of XIAP in SKOV3 ovarian cancer cells. CDDP treatment enhanced the cleavage of XIAP by HtrA1. Immuno-precipitation assay revealed that HtrA1 binds to XIAP directly both in vitro and in vivo. Increased cell resistance to cisplatin treatment was observed in SKOV3 and TOV-21G cells after stable knockdown of HtrA1 with shRNA. In contrast, over-expression of HtrA1 in OV202 cells promotes cell sensitivity to cisplatin treatment which can be reversed by increased expression of XIAP. In conclusion, our experiments reveal XIAP as a novel substrate of HtrA1 and the mechanism by which HtrA1 confers cell sensitivity to cisplatin treatment may be partially through its degradation of XIAP. 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 1531. doi:10.1158/1538-7445.AM2011-1531