Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.

We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.

Coiled-Coil and C2 Domain-Containing Protein 1A (CC2D1A) Promotes Chemotherapy Resistance in Ovarian 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.

Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers.

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.

Quinacrine upregulates p21/p27 independent of p53 through autophagy-mediated downregulation of p62-Skp2 axis 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.

Quinacrine in endometrial cancer: Repurposing an old antimalarial drug.

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.

Loss of HSulf-1: The Missing Link between Autophagy and Lipid Droplets in Ovarian Cancer.

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.

Suppression of endogenous lipogenesis induces reversion of the malignant phenotype and normalized differentiation in breast cancer.

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.

Quinacrine promotes autophagic cell death and chemosensitivity in ovarian cancer and attenuates tumor growth.

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.

HSulf-1 deficiency dictates a metabolic reprograming of glycolysis and TCA cycle in 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.

Loss of HSulf-1 promotes altered lipid metabolism in ovarian 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 Role of Heparanase and Sulfatases in the Modification of Heparan Sulfate Proteoglycans within the Tumor Microenvironment and Opportunities for Novel Cancer Therapeutics.

Heparan sulfate proteoglycans (HSPGs) are an integral and dynamic part of normal tissue architecture at the cell surface and within the extracellular matrix. The modification of HSPGs in the tumor 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 characterized 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, signaling, invasion, and metastasis. The activity or expression of these enzymes has 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 tumor initiation and progression exemplify their importance in the development and progression of cancer. Thus, as the influence of the tumor microenvironment in cancer progression becomes more apparent, the focus on targeting enzymes that degrade HSPGs highlights one approach to maintain normal tissue architecture, inhibit tumor progression, and block metastasis. This review discusses the role of these enzymes in the context of the tumor microenvironment and their promise as therapeutic targets for the treatment of cancer.

Loss of HSulf-1 expression enhances tumorigenicity by inhibiting Bim expression in ovarian cancer.

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.

Erratum to: Loss of HSulf-1 promotes altered lipid metabolism in ovarian cancer.

[This corrects the article DOI: 10.1186/2049-3002-2-13.].

Heregulin in breast cancer: old story, new paradigm.

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.

Matrix detachment and proteasomal inhibitors diminish Sulf-2 expression in breast cancer cell lines and mouse xenografts.

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.

Role of heparan sulfatases in ovarian and breast cancer.

Endosulfatases HSulf-1 and -2 (also referred to as Sulf1 and -2) represent a family of enzymes that modulate heparin binding growth factor signaling. Heparan sulfatase 1 (HSulf-1) and heparan sulfatase 2 (HSulf-2) are two important 6-O endosulfatases which remove or edit 6-O sulfate residues of N-glucosamine present on highly sulfated HS. Alteration of heparan sulfatases have been identified in the context of several cancer types. Many cancer types either exhibit increased or decreased HSulfs expression at the transcript levels. Specifically, HSulf-1 was found to be downregulated in early-stage ovarian tumors, hepatocellular carcinoma, and metastatic breast cancer patients. HSulf-2 was found to be upregulated in ductal carcinoma in situ and invasive ductal carcinoma, whereas limited information is present about HSulf-2 expression in different stages of ovarian cancers. Here, we review the important role of these sulfatases play in ovarian and breast cancers in terms of tumorigenesis such as angiogenesis, chemoresistance, apoptosis, growth factor signaling, hypoxia and metastasis. These recent discoveries have added significant understanding about these sulfate editing enzymes.

Silencing of HSulf-2 expression in MCF10DCIS.com cells attenuate ductal carcinoma in situ progression to invasive ductal carcinoma in vivo.

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.

HtrA1 sensitizes ovarian cancer cells to cisplatin-induced cytotoxicity by targeting XIAP for degradation.

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, previously we identified X-linked inhibitor of apoptosis protein (XIAP), a member of the inhibitor of apoptosis proteins family, as a potential substrate of HtrA1. The aim of our 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, coimmunoprecipitation 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, overexpression of HtrA1 in OV202 cells promoted cell sensitivity to cisplatin-induced apoptosis that 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.

Hypoxia negatively regulates heparan sulfatase 2 expression in renal cancer cell lines.

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.