H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment.

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H+-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H+-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H+-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of Km = 1.387 ±â€¯0.1674 mM Pi and Vmax = 198.6 ±â€¯10.23 Pi × h-1 × mg protein-1 hence reflecting a low affinity Pi transport. H+-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H+-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H+-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H+-dependent Pi transport was five-fold higher than the Na+-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H+-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na+-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.

The Ig VH complementarity-determining region 3-containing Rb9 peptide, inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor.

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the VHCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca2+ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.

Rebmab200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: a novel reagent for targeted antibody therapy of cancer.

NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.

Pulmonary alveolar microlithiasis. State-of-the-art review.

Pulmonary alveolar microlithiasis (PAM) is a rare genetic lung disease characterized by calcifications within the alveoli. Mutations in the SLC34A2 gene, which encodes a type IIb sodium-phosphate cotransporter, are responsible for this disease, leading to intra-alveolar accumulation of phosphate that favors the formation of microliths. The hallmark of this disorder is clinical-radiological dissociation, with typical imaging findings that correlate well with specific pathological findings. The long-term prognosis is poor and no treatment has been discovered to date. The aim of this review is to describe the main pathological, clinical, and imaging aspects of PAM, ranging from its genetic basis to treatment.

In silico analysis and immunohistochemical characterization of NaPi2b protein expression in ovarian carcinoma with monoclonal antibody Mx35.

INTRODUCTION: Ovarian adenocarcinoma is frequently detected at the late stage, when therapy efficacy is limited and death occurs in up to 50% of the cases. A potential novel treatment for this disease is a monoclonal antibody that recognizes phosphate transporter sodium-dependent phosphate transporter protein 2b (NaPi2b). MATERIALS AND METHODS: To better understand the expression of this protein in different histologic types of ovarian carcinomas, we immunostained 50 tumor samples with anti-NaPi2b monoclonal antibody MX35 and, in parallel, we assessed the expression of the gene encoding NaPi2b (SCL34A2) by in silico analysis of microarray data. RESULTS: Both approaches detected higher expression of NaPi2b (SCL34A2) in ovarian carcinoma than in normal tissue. Moreover, a comprehensive analysis indicates that SCL34A2 is the only gene of the several phosphate transporters genes whose expression differentiates normal from carcinoma samples, suggesting it might exert a major role in ovarian carcinomas. Immunohistochemical and mRNA expression data have also shown that 2 histologic subtypes of ovarian carcinoma express particularly high levels of NaPi2b: serous and clear cell adenocarcinomas. Serous adenocarcinomas are the most frequent, contrasting with clear cell carcinomas, rare, and with worse prognosis. CONCLUSION: This identification of subgroups of patients expressing NaPi2b may be important in selecting cohorts who most likely should be included in future clinical trials, as a recently generated humanized version of MX35 has been developed.