Mahesh C. Sharma

Inflammation, Microenvironment, and the Immune System in Cancer Progression

Since Virchow first proposed in 1863 that tumors could originate from sites of chronic inflammation, it has been well established that chronic inflammation both contributes to cancer progression and predisposes tissue to various types of cancer. Experimental, clinical, and epidemiological studies have all demonstrated the strong association between chronic inflammation and cancer, and many studies have correlated the prolonged presence of the inflammatory milieu with an increased risk for developing cancer. Proinflammatory cytokines, chemokines and adhesion molecules, which regulate the sequential recruitment of leukocytes, are frequently observed in tumor microenvironment. These early desmoplastic changes could stimulate fibroblasts and endothelial cell division and produce components for tissue remodeling and neovascularization, ultimately promoting neoplastic processes. In this review article we overview the current understanding of the role of chronic inflammation in neoangiogenesis, tumor initiation, promotion, and progression.

The Role of Annexin II in Angiogenesis and Tumor Progression : A Potential Therapeutic Target

It is well established that human tumors overproduce plasmin a serine protease that is known to promote angiogenesis, tumor growth and metastasis. However, the mechanism by which endothelial or tumor cells regulate the proteolytic activity of plasmin is not well understood. Cell surface receptors regulate activation of plasminogen to plasmin and its proteolytic activity. Annexin II is one of the well studied receptors for plasminogen and tPA, which binds to plasminogen and converts it to plasmin. Plasmin is a highly reactive enzyme which is physiologically involved in fibrinolysis. Since the proteolytic activity of plasmin is very tightly regulated, uncontrolled production of plasmin can degrade extracellular matrix (ECM) and basement membrane (BM) of the surrounding blood vessels. Thus plasmin plays an important role in neoangiogenesis and cancer invasion and metastasis. Therefore, the receptor which regulates plasmin generation may be an attractive target for the development of anti-cancer/anti-metastatic agents. Angiostatin (AS), internal fragment of plasminogen, has been reported to inhibit human tumor growth and metastasis. We have shown that AS binds to endothelial/cancer cell surface annexin II with high affinity and interferes with plasmin generation suggesting that the role of plasmin/plasminogen system may be more complex than we previously thought. In this review we provide a comprehensive analysis of the literature in context of the role of annexin II in angiogenesis, tumor progression and metastasis. Compelling evidence from the literature and our own findings suggest that annexin II may be a potential target for the development of effective therapeutic strategies for the treatment of cancer and its induced metastasis.

Breast cancer cell surface annexin II induces cell migration and neoangiogenesis via tPA dependent plasmin generation

Annexin II, an abundant phospholipids binding cell surface protein, binds tPA and functions as a regulator of fibrinolysis. Annexin II also mediates angiogenesis and enhances tumor growth and metastasis. However, the mechanism supporting this role is not known. Using human breast cancer model we show that invasive human breast cancer cells (MDA-MB231) synthesize annexin II and tissue plasminogen activator (tPA). In vitro both annexin II and tPA interacts which in turn convert zymogen plasminogen to reactive enzyme plasmin. Cell surface produced plasmin inhibited the migration of MDA-MB231 cells. Silencing of annexin II gene in MDA-MB231 cells abolished tPA binding therefore inhibited tPA dependent plasmin generation. These annexin II suppressed MDA-MB231 cells showed reduced motility. Immunohistochemical analysis of prediagnosed clinical specimens showed abundant secretion of tPA and expression of annexin II on the surface of invasive human breast cancer cells which correlates with neovascularization of the tumor. Taken together, these data indicate that annexin II may regulate localized plasmin generation in breast cancer. This may be an early event switching breast cancer from the prevascular phase to the vascular phase and thus contributing to aggressive cancer with the possibility of metastasis. The data provide a mechanism explaining the role of annexin II in breast cancer progression and suggest that annexin II may be an attractive target for therapeutic strategies aimed to inhibit angiogenesis and breast cancer.

Antibody-directed neutralization of annexin II (ANX II) inhibits neoangiogenesis and human breast tumor growth in a xenograft model

Activation of the fibrinolytic pathway has long been associated with human breast cancer. Plasmin is the major end product of the fibrinolytic pathway and is critical for normal physiological functions. The mechanism by which plasmin is generated in breast cancer is not yet fully described. We previously identified annexin II (ANX II), a fibrinolytic receptor, in human breast tumor tissue samples and observed a strong positive correlation with advanced stage cancer (Sharma et al., 2006a). We further demonstrated that tissue plasminogen activator (tPA) binds to ANX II in invasive breast cancer MDA-MB231cells, which leads to plasmin generation (Sharma et al., 2010). We hypothesize that ANX II-dependent plasmin generation in breast tumor is necessary to trigger the switch to neoangiogenesis, thereby stimulating a more aggressive cancer phenotype. Our immunohistochemical studies of human breast tumor tissues provide compelling evidence of a strong positive correlation between ANX II expression and neoangiogenesis, and suggest that ANX II is a potential target to slow or inhibit breast tumor growth by inhibiting neoangiogenesis. We now report that administration of anti-ANX II antibody potently inhibits the growth of human breast tumor in a xenograft model. Inhibition of tumor growth is at least partly due to attenuation of neoangiogenic activity within the tumor. In vitro studies demonstrate that anti-ANX II antibody inhibits angiogenesis on three dimensional matrigel cultures by eliciting endothelial cell (EC) death likely due to apoptosis. Taken together, these data suggest that selective disruption of the fibrinolytic activity of ANX II may provide a novel strategy for specific inhibition of neoangiogenesis in human breast cancer.

Purification and characterization of constituent androstenedione 15α-hydroxylase (Cytochrome P45015αAD) from mouse liver: Sex- and tissue-dependent expression☆

Abstract Hepatic microsomal androstenedione 15α-hydroxylase (i.e. cytochrome P450 15αAD ) was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-Sephacel, and hydroxylapatite columns at heme absorbing 417 nm, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15α-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450 15αAD , exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7α- and 15α-hydroxylation of androstenedione, representing, respectively, >5% and >93% of the total metabolites. Polyclonal antibodies against cytochrome P450 15αAD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7α and 15α-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450 15αAD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450 15αAD was 53,000, and the protein appeared as a single band on sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450 15αAD is a member of the murine cytochrome P450 2c subfamily.

Simultaneous Isolation of NADPH-Cytochrome P-450 Reductase and Cytochrome P-450 Using Tentacle Ion-Exchange Chromatography and Interspecies Comparison of the Reductase Activity

Using the same initial Fractogel (tentacle) ion-exchange chromatography to isolate murine cytochrome P-450, mouse hepatic NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was simultaneously isolated from solubilized liver microsomes and purified on a DE-52 column to a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 77 kD, and its specific activity was 25.4 mumol.min-1.mg protein-1. Purified constitutive mouse liver NADPH-cytochrome P-450 reductase was successfully reconstituted in vitro with dilauroylphosphatidyl-choline and constitutive purified mouse testosterone 2 alpha-hydroxylase (cytochrome P-450(2)alpha) with an observed activity of 13.8 nmol.min-1.nmol P-450-1. Although the partially purified reductase obtained from the Fractogel column was contaminated by significant levels of two unidentified proteins, it was as equally effective in the reconstituted system as the DE-52-derived purified reductase. Lastly, we found that rat and mouse NADPH-cytochrome P-450 reductases were similarly effective in supporting the catalytic activity of rat cytochrome P-450 2B1, but the murine reductase was 50% more effective than the rat reductase in a reconstituted system containing mouse cytochrome P-450(2)alpha.

A Comparison of Cytochrome P450-Dependent Testosterone 2α-Hydroxylase in Rat (P450 2C11) and Mouse (P4502α)

Hepatic P450 2C11 in the rat and P4502α in the mouse are unique in being the only isoforms in their respective species with testosterone 2α-hydroxylase activity. Comparing gender differences, tissue distribution and physicochemical properties, we investigated whether this uncommon catalytic activity shared by the two isoforms is dependent upon a high degree of homology. Using additional substrates (e.g. androstenedione, hexobarbital), we observed that P4502α and P450 2C11 produced no metabolites in common. Moreover, concentrations of antisera prepared against purified P4502α that inhibited 95% of P4502α-dependent testosterone 2α-hydroxylase activity had only a minimal inhibitory effect (<20%) on P450 2C11-dependent testosterone 2α-hydroxylase and were similarly unreactive to the rat isoform isolated on Western blots. Comparison of the isoforms’ N-terminal amino acid residues and two internal peptide fragments indicated almost no sequence homology (<4%). Gender-dependent tissue expression levels of P4502α and P450 2C11 revealed additional dichotomies. Whereas hepatic P4502α was moderately female-predominant (M/F; 0.62), hepatic P450 2C11 was clearly male-specific (M/F; 32.9). Murine P4502α mRNA was equally and substantially expressed in liver, kidney and brain; by contrast earlier studies reported that rat P450 2C11 was exclusively expressed in liver. The present results indicate that the unique testosterone 2α-hydroxylase activities of P4502α and P450 2C11 are expressed by two very different proteins exhibiting minimal homology.

Abstract 570: Long-term efficacy and downstream mechanism of anti-Annexin A2 monoclonal antibody in a pre-clinical model of aggressive human breast cancer

Abstract: There are considerable direct evidences to support that calcium binding protein ANX A2 is a potential target for aggressive breast cancer. The most compelling evidence is based on the fact that ANX A2 overexpress specifically in aggressive triple negative human breast cancer (TNBC) cell lines as well as in human breast cancer tissues. Previously, we and others have demonstrated a unique role of ANX A2 in cancer invasion including breast cancer. Moreover we demonstrated that anti-ANX A2 monoclonal antibody (anti-ANX A2mAb)-mediated immunoneutralization of ANX A2 inhibited invasive human breast growth in a xenograft model. We further evaluated long-term effect of multiple treatments of anti-ANX A2mAb and its mechanism of inhibition of human breast tumor growth. We now show that three treatments of anti-ANX A2mAb showed significant inhibition of breast tumor growth in immunodeficient mice and anti-tumor response was started from day 94. After treatments, we followed tumor growth for 172 days and our results demonstrated 67% inhibition of tumor growth without detectable adverse effects. Biochemical analysis demonstrated anti-ANX A2mAb treatment caused significant inhibition of tissue plasminogen activator (tPA) synthesis in the tumor microenvironment. This led to disruption of plasmin generation that consequently inhibited activation of MMP-9 and MMP-2. These results suggest that ANX A2 plays an important role in aggressive breast tumor growth by regulating proteolytic pathway in the tumor microenvironment. ANX A2 may represent a new target for the development of therapeutics for the treatment of aggressive breast cancer. Citation Format: Mahesh C. Sharma, George P. Tuszynski, Marc R. Blackman, Meena Sharma. Long-term efficacy and downstream mechanism of anti-Annexin A2 monoclonal antibody in a pre-clinical model of aggressive human breast cancer. [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 570.

Abstract 3471: Antibody-directed neutralization of fibrinolytic receptor annexin II inhibits human breast cancer growth in a xenograft model

The fibrinolytic pathway has long been known to be associated with breast cancer. Plasmin is the major end product of fibrinolytic pathway and is critical for normal physiological functions. The mechanism(s) by which plasmin is generated in breast cancer is not yet fully understood. Previously we identified ANX II, a fibrinolytic receptor, in human breast cancer samples and observed a strong positive correlation with advanced stage of cancer. We further demonstrated that the tissue plasminogen activator (tPA) binds to ANX II in invasive breast cancer MDA-MB231 cells which at least in part responsible for plasmin generation. We hypothesized that ANX II-dependent plasmin generation in breast cancer is necessary to activate the switch for neoangiogenesis thereby promoting aggressive cancer phenotype. Our immunohistochemical studies of human breast cancer tissues demonstrate strong positive correlation between annexin II expression and neoangiogenesis. These data not only indicate a link between ANX II and neoangiogenesis but also suggest that ANX II may be a potential target to slow or inhibit breast cancer growth by inhibiting neoangiogenesis. In this report we show that administration of anti-ANX II antibody potently inhibits the growth of human breast cancer in a xenograft model. Inhibition of tumor growth was due to inhibition of neoangiogenic activity within the tumor. In vitro studies demonstrated that anti-ANX II antibody inhibited angiogenesis on three dimensional matrigel cultures. The inhibition of in vitro angiogenesis was due to endothelial cell (EC) apoptosis. Taken together, these data suggest that selective disruption of fibrinolytic activity of ANX II may be a novel strategy for inhibition of neoangiogenesis in human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3471. doi:10.1158/1538-7445.AM2011-3471

Abstract 1303: Inhibition of human breast cancer cell (MDA-MB231) migration, neoangiogensis and tumor growth by selective disruption of annexin II function: in vitro and in vivo studies

Annexin II, an abundant phospholipid binding cell surface protein, binds tissue plasminogen activator (tPA) and functions as a regulator of fibrinolysis. Annexin II also mediates angiogenesis and enhances tumor growth and metastasis. However, the mechanism supporting this role is not known. Using a human breast cancer model we found that invasive human breast cancer cells (MDA-MB231) synthesize annexin II and tPA. Annexin II interacted with tPA in vitro leading to conversion of zymogen plasminogen to the reactive enzyme plasmin. Cell surface generated plasmin inhibited the migration of MDA-MB231 cells. Silencing of the annexin II gene in mDA-MB231 cells abolished tPA binding thereby inhibiting tPA dependent plasmin generation. Moreover these annexin II suppressed MDA-MB231 cells exhibited reduced motility. Immunohistochemical analysis of prediagnosed clinical specimens showed abundant secretion of tPA and expression of annexin II on the surface of invasive cancer cells which correlates with neovascularization of the tumor. Intravenous administration of the anti-annexin II antibody to mice bearing MDA-MB231 tumors significantly inhibited tumor growth possibly due to blocked neoangiogenic activity. Taken together, these data suggest that annexin II dependent plasmin generation may be an early event switching breast cancer from the prevascular phase to the vascular phase with the possibility of metastasis. Annexin II may be an attractive target for therapeutic strategies aimed to inhibit neoangiogenesis and breast cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1303.