Zhijuan Chen

γ-Secretase and Presenilin Mediate Cleavage and Phosphorylation of Vascular Endothelial Growth Factor Receptor-1

Background: γ-Secretase regulates VEGFR1 signaling. Results: Transmembrane cleavage of VEGFR1 occurs at valine 767, and VE-PTP dephosphorylation of activated VEGFR1 requires full-length presenilin 1. Conclusion: γ-Secretase cleaves VEGFR1, and full-length presenilin is a critical adaptor molecule in the dephosphorylation of VEGFR1. Significance: A greater understanding of PEDF-mediated VEGFR1 signaling and the role of γ-secretase/presenilin in the regulation of angiogenesis is important for cell biology. We have reported previously that pigment epithelium-derived factor (PEDF) can, via γ-secretase-mediated events, inhibit VEGF-induced angiogenesis in microvascular endothelial cells by both (a) cleavage and intracellular translocation of a C-terminal fragment of VEGF receptor-1 (VEGFR1) and (b) inhibition of VEGF-induced phosphorylation of VEGFR1. Using site-direct mutagenesis and transfection of wild type and mutated receptors into endothelial cells, we showed that transmembrane cleavage of VEGFR1 occurs at valine 767 and that a switch from valine to alanine at this position prevented cleavage and formation of a VEGFR1 intracellular fragment. Using siRNA to selectively knock down protein-tyrosine phosphatases (PTPs) in endothelial cells, we demonstrated that vascular endothelial PTP is responsible for dephosphorylation of activated VEGFR1. PEDF up-regulation of full-length presenilin 1 (Fl.PS1) facilitated the association of vascular endothelial PTP and VEGFR1. Knockdown of Fl.PS1 prevented dephosphorylation of VEGFR1, whereas up-regulation of Fl.PS1 stimulated VEGFR1 dephosphorylation. Fl.PS1 associated with VEGFR1 within 15 min after PEDF treatment. In conclusion, we determined the PEDF-mediated events responsible for VEGFR1 signaling and identified full-length presenilin as a critical adaptor molecule in the dephosphorylation of VEGFR1. This greater understanding of the regulation of VEGFR1 signaling will help identify novel anti-VEGF therapeutic strategies.

Discovery of structure-based small molecular inhibitor of αB-crystallin against basal-like/triple-negative breast cancer development in vitro and in vivo.

AbstractαB-crystallin (CRYAB) is present at a high frequency in poor prognosis basal-like breast tumours, which are largely absent of oestrogen, progesterone receptors and HER2 known as triple-negative breast cancer (TNBC). CRYAB functions as a molecular chaperone to bind to and correct intracellular misfolded/unfolded proteins such as vascular endothelial growth factor (VEGF), preventing non-specific protein aggregations under the influence of the tumour microenvironment stress and/or anti-cancer treatments including bevacizumab therapy. Directly targeting CRYAB can sensitize tumour cells to chemotherapeutic agents and decrease tumour aggressiveness. However, growing evidence shows that CRYAB is a critical adaptive response element after ischemic heart disease and stroke, implying that directly targeting CRYAB might cause serious unwanted side effects. Here, we used structure-based molecular docking of CRYAB and identified a potent small molecular inhibitor, NCI-41356, which can strongly block the interaction between CRYAB and VEGF165 without affecting CRYAB levels. The disruption of the interaction between CRYAB and VEGF165 elicits in vitro anti-tumour cell proliferation and invasive effects through the down-regulation of VEGF signalling in the breast cancer cells. The observed in vitro anti-tumour angiogenesis of endothelial cells might be attributed to the down-regulation of paracrine VEGF signalling in the breast cancer cells after treatment with NCI-41356. Intraperitoneal injection of NCI-41356 greatly inhibits the tumour growth and vasculature development in in vivo human breast cancer xenograft models. Our findings provide ‘proof-of-concept’ for the development of highly specific structure-based alternative targeted therapy for the prevention and/or treatment of TNBC.

γ-Secretase Inhibition of Murine Choroidal Neovascularization Is Associated with Reduction of Superoxide and Proinflammatory Cytokines

PURPOSE This study aimed to determine whether upregulation of γ-secretase could inhibit laser-induced choroidal neovascularization (CNV) and if this was associated with a reduction in both oxidative stress and proinflammatory cytokines. METHODS γ-Secretase, or its catalytic subunit presenilin 1 (PS1), were upregulated by exposure to either pigment epithelial derived factor (PEDF) or an AAV2 vector containing a PS1 gene driven by a vascular endothelial-cadherin promoter. Retinal endothelial cells were infected with AAV2 or exposed to PEDF in the presence or absence of VEGF and in vitro angiogenesis determined. Mouse eyes either received intravitreal injection of PEDF, DAPT (a γ-secretase inhibitor) or PEDF + DAPT at the time of laser injury, or AAV2 infection 3 weeks before receiving laser burns. Lesion volume was determined 14 days post laser injury. Superoxide generation, antioxidant activity and the production of proinflammatory mediators were assessed. Knockdown of γ-secretase was achieved using siRNA. RESULTS γ-Secretase upregulation and PS1 overexpression suppressed VEGF-induced in vitro angiogenesis and in vivo laser-induced CNV. This was associated with a reduction in the expression of VEGF and angiogenin 1 together with reduced superoxide anion generation and an increase in MnSOD compared with untreated CNV eyes. PS1 overexpression reduced proinflammatory factors and microglial activation in eyes with CNV compared with control. siRNA inhibition of γ-secretase resulted in increased angiogenesis. CONCLUSIONS γ-Secretase, and in particular PS1 alone, are potent regulators of angiogenesis and this is due in part to stabilizing endogenous superoxide generation and reducing proinflammatory cytokine expression during CNV.

UFH-001 cells: A novel triple negative, CAIX-positive, human breast cancer model system

ABSTRACT Human cell lines are an important resource for research, and are often used as in vitro models of human diseases. In response to the mandate that all cells should be authenticated, we discovered that the MDA-MB-231 cells that were in use in our lab, did not validate based on the alleles of 9 different markers (STR Profile). We had been using this line as a model of triple negative breast cancer (TNBC) that has the ability to form tumors in immuno-compromised mice. Based on marker analysis, these cells most closely resembled the MCF10A line, which are a near diploid and normal mammary epithelial line. Yet, the original cells express carbonic anhydrase IX (CAIX) both constitutively and in response to hypoxia and are features that likely drive the aggressive nature of these cells. Thus, we sought to sub-purify CAIX-expressing cells using Fluorescence Activated Cell Sorting (FACS). These studies have revealed a new line of cells that we have name UFH-001, which have the TNBC phenotype, are positive for CAIX expression, both constitutively and in response to hypoxia, and behave aggressively in vivo. These cells may be useful for exploring mechanisms that underlie progression, migration, and metastasis of this phenotype. In addition, constitutive expression of CAIX allows its evaluation as a therapeutic target, both in vivo and in vitro.

Selective inhibition of CA IX over CA XII using benzene sulfonamides: Disconnect between CA activity and growth inhibition in breast cancer cells

Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membrane-bound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.Carbonic anhydrases (CAs) have been implicated in tumor progression particularly membrane bound CA isoform IX (CA IX). In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with a better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The affinity of these inhibitors are two to three orders of magnitude lower in breast cancer cells, that selectively express CA IX or CA XII, relative to recombinant CA proteins. However, we show significantly greater inhibition of CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells in which CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth inhibition.

Differential expression and function of CAIX and CAXII in breast cancer: A comparison between tumorgraft models and cells

Carbonic anhydrase IX (CAIX) and XII (CAXII) are transmembrane proteins that are associated with cancer progression. We have previously described the catalytic properties of CAIX in MDA-MB-231 breast cancer cells, a line of cells that were derived from a patient with triple negative breast cancer. We chose this line because CAIX expression in breast cancer is a marker of hypoxia and a prognosticator for reduced survival. However, CAXII expression is associated with better survival statistics than those patients with low CAXII expression. Yet CAIX and CAXII have similar catalytic activities. Here we compare the potential roles of CAIX and CAXII in the context of TNBC and estrogen receptor (ER)-positive breast cancer. In tumor graft models, we show that CAIX and CAXII exhibit distinct expression patterns and non-overlapping. We find the same pattern across a panel of TNBC and luminal breast cancer cell lines. This affords an opportunity to compare directly CAIX and CAXII function. Our data suggest that CAIX expression is associated with growth potentiation in the tumor graft model and in a TNBC line using knockdown strategies and blocking activity with an impermeant sulfonamide inhibitor, N-3500. CAXII was not associated with growth potentiation. The catalytic activities of both CAIX and CAXII were sensitive to inhibition by N-3500 and activated at low pH. However, pH titration of activity in membrane ghosts revealed significant differences in the catalytic efficiency and pKa values. These features provide evidence that CAIX is a more efficient enzyme than CAXII at low pH and that CAIX shifts the equilibrium between CO2 and bicarbonate in favor of CO2 production by consuming protons. This suggests that in the acidic microenvironment of tumors, CAIX plays a role in stabilizing pH at a value that favors cancer cell survival.

Abstract B25: Characterization, targeting, and modulation of carbonic anhydrase IX activity for the development of small-molecule inhibitors to treat triple-negative breast cancer

The microenvironment within a solid tumor is usually heterogeneous with certain regions being acidic and hypoxic. These acidic and hypoxic regions arise from rapidly proliferating cells combined with poor tumor perfusion. Cancer cells cope with these hostile changes in their microenvironment by expressing genes that are essential for survival. One of the coping mechanisms is an upregulation of pH regulatory factors, including carbonic anhydrase IX (CAIX). The action of CAIX helps to maintain physiological pH inside the cell (pHi) while regulating extracellular acidification (pHe). Extracellular acidification of the tumor microenvironment promotes local invasion, metastasis and decreases the effectiveness of adjuvant therapies, thus contributing to poor clinical outcome. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in a triple negative breast cancer cell line. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors using X-ray crystallography methods. These structures show the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity measured as 18O exchange between H2O16 and H13C18O3 . We have also investigated the effect CAIX inhibition on cancer cell metabolism and extracellular acidification using Seahorse technology. This reveals that CAIX may contribute to the “non-glycolytic” acidification process. In total, these observations indicate that CAIX is a viable small molecular drug target and contribute to our understanding of the function of CAIX in modulating pH in cancer cells. Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Nicole Lamas, Shingkuang Tu, Fabrizio Carta, Claudiu T. Superan, Robert McKenna, Susan C. Frost. Characterization, targeting, and modulation of carbonic anhydrase IX activity for the development of small-molecule inhibitors to treat triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B25.

Abstract C37: Differential expression of carbonic anhydrases and proton transporters in co-cultured models of the breast cancer microenvironment

Extracellular acidification (pHe) within the tumor microenvironment is a well-accepted hallmark for aggressive tumor behavior. Hypoxia is thought to play a key role in the acidic tumor microenvironment. Hypoxia triggers a shift to glycolytic metabolism and leads to an excess of acidic products, including lactic acid, protons and carbon dioxide. To counteract the acidosis, cells activate the pH regulatory machinery which has two major transport arms: the lactate and proton export arm and bicarbonate import arm. These two arms operate through several types of transmembrane proton transporters (TPTs). These transporters include the monocarboxylate transporters (MCTs), the vacuolar ATPase (v-ATPase), the sodium/proton exchangers (NHE-1), and the sodium/bicarbonate transporters (NBC). In addition, it is hypothesized that membrane-bound isoforms of the carbonic anhydrase family (CAIX and CAXII) may balance pHe to the advantage of cancer cells by improved buffering utilizing the CO2/bicarbonate system. Within the tumor microenvironment, there are both malignant cells and adjacent stromal cells, including fibroblasts, endothelial cells, adipocytes, pericytes, and inflammatory macrophages. Communication and interactions between tumor cells and stromal cells may contribute to the pH regulation of the tumor microenvironment. A recent study by Fiaschi et al. (2013) showed that cultured, healthy human prostate fibroblasts could be converted to the phenotype of cancer-associated fibroblasts (CAFs) by incubation with conditioned medium from cultured prostate cancer cells. This conversion induced the upregulation of CAIX. This might suggest that CAFs play a role in pH regulation in the tumor microenvironment. To determine if breast cancer fibroblasts respond in like, we co-cultured MDA-MB-231 (triple-negative, CAIX-positive) and T47D (ER-positive, CAXII-positive) breast cancer cells with human, mammary-specific, cancer-associated fibroblasts using the transwell system. In this setting, we were unable to demonstrate that either CAIX or CAXII expression changed in the CAFs. We also explored the expression of the TPTs. Interestingly, triple negative and ER-positive breast cancer cells showed differential expression of these family members. In the co-culture system, vATPase expression increased in CAFs co-cultured with the MDA cells where reduced expression was observed. Knockdown of CAIX in the MDA cells did not alter this difference. Yet loss of CAIX in the MDA cells reduced their expression of NHE-1. We observed no expression difference in CAFs co-cultured with T47D cells. Knockdown of CAXII in T47D cells enhanced NHE-1 expression but reduced MCT4 expression. These preliminary data suggest that there are complicated interactions between CAFs and breast cancer cells not related to CAIX or CAXII expression. Yet, loss of expression of CAIX or CAXII did change the expression of TPTs in the cancer cells, suggesting the existence of compensatory mechanisms. Citation Format: Zhijuan Chen, Susan C. Frost. Differential expression of carbonic anhydrases and proton transporters in co-cultured models of the breast cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C37.

Abstract LB-4: Mechanisms by which the unfolded protein response/α-Basic-crystallin (CRYAB) regulates VEGF signaling of tumor endothelial cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Angiogenesis is an essential component of tumour development and is tightly regulated by a network of pro-angiogenic and anti-angiogenic factors. The tumour cells are known to produce many of the proteins that interact with vascular endothelial cells and drive the angiogenic process. Of the known angiogenic factors, VEGF has been established as a potent inducer of tumor angiogenesis. Like almost all secretory and membrane proteins, VEGF attains its final folded or assembly conformation in ER before making its way to its target organelles or the cell surface. The deleterious tumor microenvironment characterized by progressive hypoxia, nutrient starvation and acidosis results in the ER stress in the tumor vascular endothelial cells, which in turn leads to the unfolded/misfolded VEGF accumulated in the ER. Molecular chaperon proteins counteract the formation of aberrantly folded VEGF and allow its correct folding. One of molecular chaperons is CRYAB. It has been reported that CRYAB contains interactive sequences for VEGF. The misfolded VEGF interacts with BiP (chaperone immunoglobulin heavy chain-binding protein) causing its release from one or more of the three ER stress sensors [PKR-like ER kinase (PERK), inositol-requiring protein-1 (IRE1) and transcription factor 6 (ATF6)], which leads to activation of the unfolded protein response (UPR) pathway. All three sensor pathways can lead to chaperone induction but the IRE1 and ATF6 pathways are thought to play the major role in up-regulation of CRYAB. Activation of IRE1 generates a mature XBP-1 mRNA by unconventional splicing of XBP-1 pre-mRNA and activated XBP-1 up-regulates CRYAB. Activation of ATF6 results in its translocation to the Golgi apparatus and proteolytic cleavage to yield its cytosolic domain not only for directly up-regulating genes encoding CRYAB and folding catalysts, but also for assisting IRE1-dependent upregulation of CRYAB. An excessively elevated level of CRYAB has been detected in breast carcinomas, correlating with lymph node involvement resulting in metastasis and short term survival. Taken together, it is clear that CRYAB has the potential to a) protect VEGF from intracellular degradation thus maintaining the intracellular pool and b) increase VEGF secretion. Both these actions serve to enhance the autocrine VEGF signaling response which serves as one of strategies adapted by the tumor vasculature to perpetuate aberrant angiogenesis. 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 LB-4. doi:10.1158/1538-7445.AM2011-LB-4