Safaa A. Ibrahim

Breast cancer associated a2 isoform vacuolar ATPase immunomodulates neutrophils: potential role in tumor progression.

In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy.

Monte Carlo simulation analysis of ceftobiprole, dalbavancin, daptomycin, tigecycline, linezolid and vancomycin pharmacodynamics against intensive care unit-isolated methicillin-resistant Staphylococcus aureus

The aim of the present study was to compare the potential of ceftobiprole, dalbavancin, daptomycin, tigecycline, linezolid and vancomycin to achieve their requisite pharmacokinetic/pharmacodynamic (PK/PD) targets against methicillin‐resistant Staphylococcus aureus isolates collected from intensive care unit (ICU) settings. Monte Carlo simulations were carried out to simulate the PK/PD indices of the investigated antimicrobials. The probability of target attainment (PTA) was estimated at minimum inhibitory concentration values ranging from 0.03 to 32 μg/mL to define the PK/PD susceptibility breakpoints. The cumulative fraction of response (CFR) was computed using minimum inhibitory concentration data from the Canadian National Intensive Care Unit study. Analysis of the simulation results suggested the breakpoints of 4 μg/mL for ceftobiprole (500 mg/2 h t.i.d.), 0.25 μg/mL for dalbavancin (1000 mg), 0.12 μg/mL for daptomycin (4 mg/kg q.d. and 6 mg/kg q.d.) and tigecycline (50 mg b.i.d.), and 2 μg/mL for linezolid (600 mg b.i.d.) and vancomycin (1 g b.i.d. and 1.5 g b.i.d.). The estimated CFR were 100, 100, 70.6, 88.8, 96.5, 82.4, 89.4, and 98.3% for ceftobiprole, dalbavancin, daptomycin (4 mg/kg/day), daptomycin (6 mg/kg/day), linezolid, tigecycline, vancomycin (1 g b.i.d.) and vancomycin (1.5 g b.i.d.), respectively. In conclusion, ceftobiprole and dalbavancin have the highest probability of achieving their requisite PK/PD targets against methicillin‐resistant Staphylococcus aureus isolated from ICU settings. The susceptibility predictions suggested a reduction of the vancomycin breakpoint to 1 μg/mL.

Pregnancy is a model for tumors, not transplantation

Nearly 65 years have passed since Peter Medawar posed the following question: “How does the pregnant mother contrive to nourish within itself, for many weeks or months, a fetus that is an antigenically foreign body.” Now, understanding of reproductive immunology has demonstrated that the HLA antigens in the placenta are non‐classical and do not induce rejection. In the placenta and in tumors, 50% or more of the cells are cells of the immune system and were once thought to be primed and ready for killing tumors or the “fetal transplant” but these cells are not potential killers but abet the growth of either the tumor or the placenta. We believe that these cells are there to create an environment, which enhances either placental or tumor growth. By examining the similarities of the placentas and tumors immune cells, novel mechanisms to cause tumors to be eliminated can be devised.

Selective inhibition of tumor cell associated Vacuolar-ATPase ‘a2’ isoform overcomes cisplatin resistance in ovarian cancer cells

Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo‐resistance. These pH alterations are mediated by aberrant activation of key multi‐subunit proton pumps, Vacuolar‐ATPases (V‐ATPases). In tumor cells, its ‘a2’ isoform (V‐ATPase‐V0a2) is a component of functional plasma–membrane complex and promotes tumor invasion through tumor‐acidification and immuno‐modulation. Its involvement in chemo‐resistance has not been studied. Here, we show that V‐ATPase‐V0a2 is over‐expressed in acquired‐cisplatin resistant OVCA cells (cis‐A2780/cis‐TOV112D). Of all the ‘a’ subunit isoforms, V‐ATPase‐V0a2 exhibited an elevated expression on plasma membrane of cisplatin‐resistant cells compared to sensitive counterparts. Immuno‐histochemistry revealed V‐ATPase‐V0a2 expression in both low grade (highly drug‐resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V‐ATPase‐V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin‐DNA‐adduct formation and suppressed DNA‐repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum‐associated DNA‐damage. As an indicator of reduced metastasis and chemo‐resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2‐knockdown resistant cells. Interestingly, pre‐treatment with monoclonal V0a2‐inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V‐ATPase‐V0a2 could serve as a therapeutic strategy for chemo‐resistant ovarian carcinoma and improve efficacy of platinum drugs.

Mammary epithelium‐specific inactivation of V‐ATPase reduces stiffness of extracellular matrix and enhances metastasis of breast cancer

Extracellular matrix (ECM) critically impacts tumor progression and is influenced by both cancer and host tissue cells. While our understanding of cancer cell ECM remodeling is widespread, the importance of host tissue ECM, which provides initial congenial environment for primary tumor formation, is partly understood. Here, we report a novel role of epithelial cell‐associated vacuolar ATPase ‘a2’ isoform (a2V) in regulating breast tissue ECM stiffness to control metastasis. Using a mammary gland‐specific a2V‐knockout model, we show that in the absence of a2V, breast tumors exhibit atypically soft tumor phenotype, less tumor rigidity, and necrotic tumor microenvironment. These tumors contain a decreased number of cancer cells at primary tumor site, but showed extensive metastases compared to control. Nanomechanical evaluation of normal breast tissues revealed a decrease in stiffness and collagen content in ECM of a2V‐deleted breast tissues. Mechanistically, inhibition of a2V expression caused dispersed Golgi morphology with relocation of glycosyltransferase enzymes to early endosomes in mammary epithelial cells. This resulted in defective glycosylation of ECM proteins and production of compromised ECM that further influenced tumor metastasis. Clinically, in patients with cancer, low a2V expression levels in normal breast tissue correlated with lymph node metastasis. Thus, using a new knockout mouse model, we have identified a2V expression in epithelial cells as a key requirement for proper ECM formation in breast tissue and its expression levels can significantly modulate breast tumor dissemination. Evaluation of a2V expression in normal breast tissues can help in identifying patients with high risk of developing metastases.

Cancer derived peptide of vacuolar ATPase ‘a2’ isoform promotes neutrophil migration by autocrine secretion of IL-8

Neutrophils play significant regulatory roles within the tumor microenvironment by directly promoting tumor progression that leads to poor clinical outcomes. Identifying the tumor associated molecules that regulate neutrophil infiltration into tumors may provide new and specific therapeutic targets for cancer treatment. The a2-isoform of vacuolar ATPase (a2V) is uniquely and highly expressed on cancer cell plasma membrane. Cancer cells secrete a peptide from a2V (a2NTD) that promotes the pro-tumorigenic properties of neutrophils. This provides a2V the propensity to control neutrophil migration. Here, we report that the treatment of human neutrophils with recombinant a2NTD leads to neutrophil adherence and polarization. Moreover, a2NTD treatment activates surface adhesion receptors, as well as FAK and Src kinases that are essential regulators of the migration process in neutrophils. Functional analysis reveals that a2NTD can act as a chemo-attractant and promotes neutrophil migration. In addition, a2Neuɸ secrete high levels of IL-8 via NF-κB pathway activation. Confirmatory assays demonstrate that the promoted migration of a2Neuɸ was dependent on the autocrine secretion of IL-8 from a2Neuɸ. These findings demonstrate for the first time the direct regulatory role of cancer associated a2-isoform V-ATPase on neutrophil migration, suggesting a2V as a potential target for cancer therapy.

Hematopoietic stem cell specific V-ATPase controls breast cancer progression and metastasis via cytotoxic T cells

The interaction of recruited immune effector cells and cancer cells within tumor microenvironment (TME) shapes the fate of cancer progression and metastasis. Many cancers including breast cancer, express a specific vacuolar ATPase (a2V) on their cell surface which acidifies the extracellular milieu helping cancer cell proliferation and metastasis. To understand the role of immune cell-associated-a2V during breast tumor pathogenesis, we knocked-out a2V (KO) from the hematopoietic stem cells (HSC) and generated breast tumors in mice. The a2V-KO mice developed faster growing, larger, and metastatic breast tumors compared to control mice. Further investigation of the TME revealed a significant reduction in the presence of CD4+ and CD8+ T cells in the a2V-KO tumors. Targeted RNA-Seq of the cells of the TME demonstrated that pro-inflammatory cytokines, death receptors, death receptor ligands, and cytotoxic effectors were significantly down-regulated within the a2V-KO TME. Interestingly, analysis of immune cells in the blood, spleen, and thymus of the non-tumor bearing a2V-KO mice revealed a significant decrease in CD4+ and CD8+ T cell populations. For the first time, this study demonstrates that inhibition of V-ATPase expression in HSC leads to a decrease in CD4+ and CD8+ T cell populations and thus promotes breast tumor growth and metastasis.

Microtubule inhibitor, SP-6-27 inhibits angiogenesis and induces apoptosis in ovarian cancer cells

In ovarian cancer (OVCA), treatment failure due to chemo-resistance is a serious challenge. It is therefore critical to identify new therapies that are effective against resistant tumors and have reduced side effects. We recently identified 4-H-chromenes as tubulin depolymerizing agents that bind to colchicine site of beta-tubulin. Here, we screened a chemical library of substituted 4-H-chromenes and identified SP-6-27 to exhibit most potent anti-proliferative activity towards a panel of human cisplatin sensitive and resistant OVCA cell lines with 50% inhibitory concentration (IC50; mean ± SD) ranging from 0.10 ± 0.01 to 0.84 ± 0.20 μM. SP-6-27 exhibited minimum cytotoxicity to normal ovarian epithelia. A pronounced decrease in microtubule density as well as G2/M cell cycle arrest was observed in SP-6-27 treated cisplatin sensitive/resistant OVCA cells. The molecular mechanism of SP-6-27 induced cell death revealed modulation in cell-cycle regulation by upregulation of growth arrest and DNA damage inducible alpha transcripts (GADD45). An enhanced intrinsic apoptosis was observed in OVCA cells through upregulation of Bax, Apaf-1, caspase-6, -9, and caspase-3. In vitro wound healing assay revealed reduced OVCA cell migration upon SP-6-27 treatment. Additionally, SP-6-27 and cisplatin combinatorial treatment showed enhanced cytotoxicity in chemo-sensitive/resistant OVCA cells. Besides effect on cancer cells, SP-6-27 further restrained angiogenesis by inhibiting capillary tube formation by human umbilical vein endothelial cells (HUVEC). Together, these findings show that the chromene analog SP-6-27 is a novel chemotherapeutic agent that offers important advantages for the treatment of ovarian cancer.In ovarian cancer (OVCA), treatment failure due to chemo-resistance is a serious challenge. It is therefore critical to identify new therapies that are effective against resistant tumors and have reduced side effects. We recently identified 4-H-chromenes as tubulin depolymerizing agents that bind to colchicine site of beta-tubulin. Here, we screened a chemical library of substituted 4-H-chromenes and identified SP-6-27 to exhibit most potent anti-proliferative activity towards a panel of human cisplatin sensitive and resistant OVCA cell lines with 50% inhibitory concentration (IC50; mean ± SD) ranging from 0.10 ± 0.01 to 0.84 ± 0.20 μM. SP-6-27 exhibited minimum cytotoxicity to normal ovarian epithelia. A pronounced decrease in microtubule density as well as G2/M cell cycle arrest was observed in SP-6-27 treated cisplatin sensitive/resistant OVCA cells. The molecular mechanism of SP-6-27 induced cell death revealed modulation in cell-cycle regulation by upregulation of growth arrest and DNA damage inducible alpha transcripts (GADD45). An enhanced intrinsic apoptosis was observed in OVCA cells through upregulation of Bax, Apaf-1, caspase-6, -9, and caspase-3.In vitro wound healing assay revealed reduced OVCA cell migration upon SP-6-27 treatment. Additionally, SP-6-27 and cisplatin combinatorial treatment showed enhanced cytotoxicity in chemo-sensitive/resistant OVCA cells. Besides effect on cancer cells, SP-6-27 further restrained angiogenesis by inhibiting capillary tube formation by human umbilical vein endothelial cells (HUVEC). Together, these findings show that the chromene analog SP-6-27 is a novel chemotherapeutic agent that offers important advantages for the treatment of ovarian cancer.

Abstract 1231: Novel chromene analogs as small-molecule microtubule destabilizers for the treatment of chemo-resistant ovarian cancer

Ovarian cancer is the most lethal gynecological malignancy with a 5-year survival rate of less than 40%, primarily because of treatment failure due to emergence of chemo-resistance to standard agents. This indicates a dire need for new treatments to improve survival rates. The tubulin dynamics is a promising target for new chemotherapeutic agents. In continuation of our efforts, using medicinal chemistry approach, we recently identified small molecular inhibitors of tubulin polymerization called chromenes, as new ovarian anti-cancer agents. A set of chromene compounds were screened for anti-cancer effect on cisplatin sensitive (A2780, SKOV-3, TOV112D) and cisplatin resistant (OVCAR-3, A2780-cisR, TOV112D-cisR) ovarian cancer cells. In vitro cell viability was assessed by fluorescence based Alamar Blue assay. Our preliminary studies identified SP-6-27 (IC50 range: 0.102± 0.006 - 2.24±0.056 μM) and SP-6-37 (IC50 range: 0.258± 0.159 - 3.61±0.049 μM) as most potent chromene analogs. Both compounds exhibited a potent anticancer activity towards both cisplatin sensitive (SP-6-27 IC50: 0.14± 0.03 μM; SP-6-37 IC50: 1.35± 0.88 μM) and cisplatin resistant (SP-6-27 IC50: 0.81± 1.2 μM; SP-6-37 IC50: 2.09± 1.7 μM) cell lines. The compounds exhibited least cytotoxicity towards normal ovarian epithelial cells (SP-6-27- IC50: 83.35 ± 9.47 μM; SP-6-37- IC50: 75.77 ± 5.37 μM). Additionally, the analysis of apoptotic changes as determined by Annexin-V assay revealed an enhanced apoptosis in both cisplatin sensitive and resistant cells upon treatment with compounds SP-6-27 or SP-6-37. Together, the data demonstrates that ovarian cancer cell lines respond sensitively to SP-6-27 and SP-6-37, demonstrating that the chromene scaffold is effective in suppressing ovarian cancer cell growth. The findings indicate that the novel chromene analogs are a new class of chemotherapeutic agents that will offer advantages for the treatment of ovarian cancer. Citation Format: Arpita Kulshrestha, Safaa A. Ibrahim, Gajendra K. Katara, Renukadevi Patil, Shivaputra Patil, Kenneth D. Beaman. Novel chromene analogs as small-molecule microtubule destabilizers for the treatment of chemo-resistant ovarian 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 1231.

Abstract 4939: The baffling effect of bafilomycin on Notch signaling in triple-negative breast cancer

Triple Negative Breast Cancers (TNBC) constitute 15 to 20% of all breast cancers and lack expression of ER/PR and Her2 receptors. Owing to an aggressive phenotype and lack of targeted therapies, TNBC has poor prognosis. Notch signaling is an evolutionarily conserved signaling pathway known to be important for normal mammary development and breast cancer progression. Although there has been considerable research on the role of Notch in TNBC, little is known about its regulation. Identifying novel molecules that regulate Notch can lead to the development of effective combinatorial treatments. The Vacuolar ATPase (V-ATPase) is a proton pump responsible for maintaining pH. Expression of V-ATPase is upregulated in tumor cells, promoting cancer proliferation and metastasis. We have previously identified the role of V-ATPase a2 isoform (V0a2) in pregnancy and cancer. Here, we report that V-ATPase regulates Notch Signaling in TNBC. Upon treatment with Bafilomycin A1 (BafA1), a specific V-ATPase inhibitor, Notch and Wnt Signaling genes were significantly upregulated in TNBC cell lines MDA-MB-231 and MDA-MB-468 (p Citation Format: Sahithi Pamarthy, Mukesh K. Jaiswal, Arpita Kulshrestha, Gajendra Katara, Safaa Ibrahim, Alice Gilman-Sachs, Kenneth D. Beaman. The baffling effect of bafilomycin on Notch signaling in triple-negative breast cancer. [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 4939. doi:10.1158/1538-7445.AM2015-4939