Sahithi Pamarthy

Notch Signaling in Inflammation-Induced Preterm Labor

Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo, Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages, and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1β) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses, and its inhibition improves in-utero survival of live fetuses.

Inhibition of vacuolar ATPase subunit in tumor cells delays tumor growth by decreasing the essential macrophage population in the tumor microenvironment

In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma as well as vesicular membranes and critically influences metastatic behavior. The soluble, cleaved N-terminal domain of V-ATPase a2 isoform is associated with in vitro induction of tumorigenic characteristics in macrophages. This activity led us to further investigate its in vivo role in cancer progression by inhibition of a2 isoform (a2V) in tumor cells and the concomitant effect on tumor microenvironment in the mouse 4T-1 breast cancer model. Results showed that macrophages cocultivated with a2V knockdown (sh-a2) 4T-1 cells produce lower amounts of tumorigenic factors in vitro and have reduced ability to suppress T-cell activation and proliferation compared with control 4T-1 cells. Data analysis showed a delayed mammary tumor growth in Balb/c mice inoculated with sh-a2 4T-1 cells compared with control. The purified CD11b+ macrophages from sh-a2 tumors showed a reduced expression of mannose receptor-1 (CD206), interleukin-10, transforming growth factor-β, arginase-1, matrix metalloproteinase and vascular endothelial growth factor. Flow cytometric analysis of tumor-infiltrated macrophages showed a significantly low number of F4/80+CD11c+CD206+ macrophages in sh-a2 tumors compared with control. In sh-a2 tumors, most of the macrophages were F4/80+CD11c+ (antitumor M1 macrophages) suggesting it to be the reason behind delayed tumor growth. Additionally, tumor-infiltrating macrophages from sh-a2 tumors showed a reduced expression of CD206 compared with control whereas CD11c expression was unaffected. These findings demonstrate that in the absence of a2V in tumor cells, the resident macrophage population in the tumor microenvironment is altered which affects in vivo tumor growth. We suggest that by involving the host immune system, tumor growth can be controlled through targeting of a2V on tumor cells.

The Vacuolar ATPase a2-subunit regulates Notch signaling in triple-negative breast cancer cells.

Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer with poor prognosis for which no targeted therapies are currently available. Notch signaling has been implicated in breast cancer but the factors that control Notch in TNBC are unknown. Because the Vacuolar ATPase has been shown to be important in breast cancer invasiveness, we investigated the role of a2-subunit isoform of Vacuolar ATPase (a2V) in regulating Notch signaling in TNBC. Confocal microscopy revealed that among all the ‘a’ subunit isoforms, a2V was uniquely expressed on the plasma membrane of breast cancer cells. Both a2V and NOTCH1 were elevated in TNBC tumors tissues and cell lines. a2V knockdown by siRNA as well as V-ATPase inhibition by Bafilomycin A1 (Baf A1) in TNBC cell lines enhanced Notch signaling by increasing the expression of Notch1 intracellular Domain (N1ICD). V-ATPase inhibition blocked NICD degradation by disrupting autophagy and lysosomal acidification as demonstrated by accumulation of LC3B and diminished expression of LAMP1 respectively. Importantly, treatment with Baf A1 or anti-a2V, a novel-neutralizing antibody against a2V hindered cell migration of TNBC cells. Our findings indicate that a2V regulates Notch signaling through its role in endolysosomal acidification and emerges as a potential target for TNBC.

Role of Notch signaling during lipopolysaccharide-induced preterm labor.

Notch signaling pathways exert effects throughout pregnancy and are activated in response to TLR ligands. To investigate the role of Notch signaling in preterm labor, Notch receptors (Notch1–4), its ligand Delta‐like protein‐1, transcriptional repressor hairy and enhancer of split‐1, and Notch deregulator Numb were assessed. Preterm labor was initiated on gestation d 14.5 by 1 of 2 methods: 1) inflammation‐induced preterm labor: intrauterine injection of LPS (a TLR4 agonist) and 2) hormonally induced preterm labor: subcutaneous injection of mifepristone. Delta‐like protein‐1, Notch1, and hairy and enhancer of split‐1 were elevated significantly, and Numb was decreased in the uterus and placenta of inflammation‐induced preterm labor mice but remained unchanged in hormonally induced preterm labor compared with their respective controls. F4/80+ macrophage polarization was skewed in the uterus of inflammation‐induced preterm labor toward M1‐positive (CD11c+) and double‐positive [CD11c+ (M1) and CD206+ (M2)] cells. This process is dependent on activation of Notch signaling, as shown by suppression of M1 and M2 macrophage‐associated cytokines in decidual macrophages in response to γ‐secretase inhibitor (an inhibitor of Notch receptor processing) treatment ex vivo. γ‐Secretase inhibitor treatment also diminished the LPS‐induced secretion of proinflammatory cytokines and chemokines in decidual and placental cells cultured ex vivo. Furthermore, treatment with recombinant Delta‐like protein‐1 ligand enhanced the LPS‐induced proinflammatory response. Notch ligands (Jagged 1 and 2 and Delta‐like protein‐4) and vascular endothelial growth factor and its receptor involved in angiogenesis were reduced significantly in the uterus and placenta during inflammation‐induced preterm labor. These results suggest that up‐regulation of Notch‐related inflammation and down‐regulation of angiogenesis factors may be associated with inflammation‐induced preterm labor but not with hormonally induced preterm labor.

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.

Overcoming immunosuppression in bone metastases

Bone metastases are present in up to 70% of advanced prostate and breast cancers and occur at significant rates in a variety of other cancers. Bone metastases can be associated with significant morbidity. The establishment of bone metastasis activates several immunosuppressive mechanisms. Hence, understanding the tumor-bone microenvironment is crucial to inform the development of novel therapies. This review describes the current standard of care for patients with bone metastatic disease and novel treatment options targeting the microenvironment. Treatments reviewed include immunotherapies, cryoablation, and targeted therapies. Combinatorial treatment strategies including targeted therapies and immunotherapies show promise in pre-clinical and clinical studies to overcome the suppressive environment and improve treatment of bone metastases.

Male fertility and apoptosis in normal spermatogenesis are regulated by vacuolar-ATPase isoform a2.

The a2 isoform of vacuolar-ATPase (ATP6V0A2, referred to as a2V) is required for normal spermatogenesis and maturation of sperm. Treatment of male mice with anti-a2V disturbs the testicular cytokine/chemokine balance and leads to severe deficiencies of spermatogenesis. The aim of the present study was to investigate the role of a2V in male fertility and in the regulation of apoptotic pathways required for normal spermatogenesis in mice. To study the role of a2V single dose of anti-a2V monoclonal antibody or mouse IgG isotype (3μg/animal) was injected i.p. into males on alternate days for 10 days. The expression of sperm maturation-related molecules and pro-apoptotic molecules was measured by real-time PCR or immunohistochemistry in control and anti-a2V-treated testes. The caspase levels and their activity were measured by western blot and fluorometry. We found that the expression of the sperm maturation-related molecules SPAM1, ADAM1, and ADAM2 was significantly decreased in testes from anti-a2V-treated males. The expression of pro-apoptotic molecules (Bax, p53, and p21) and molecules involved in the intrinsic pathway of apoptosis (caspase-9, caspase-3, and PARP), which are crucial for normal spermatogenesis was significantly reduced in testes from anti-a2V-treated males compared with the control. The total ATP level was significantly lower in anti-a2V-treated testes. The data provide novel evidence showing that a2V can regulate the apoptotic pathways, an essential testicular feature, and is necessary for efficient spermatogenesis.

The curious case of vacuolar ATPase: regulation of signaling pathways

The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.