High expression of SLC34A2 is a favorable prognostic marker in lung adenocarcinoma patients.

Dysregulation of SLC34A2 (NaPi2b) in tumors has attracted wide attention, but its expression and function in non-small cell lung cancer remains unclear. By examining its expression in lung adenocarcinoma and correlation to patient outcome, we aimed to explore its prognostic and therapeutic values in this deadly disease. Overall, 175 cases of lung adenocarcinoma sample were included in this study. Histological subtyping of them was diagnosed according to standards of the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society in 2011. Protein expression of SLC34A2 and anaplastic lymphoma kinase in these samples was determined by immunohistochemistry. Epidermal growth factor receptor mutations were examined using amplification refractory mutation system. Statistical analysis was performed using software of Pearson's correlation coefficient. High expression of SLC34A2 was identified in about 2/3 patients and correlated with significantly better patient's overall survival. Epidermal growth factor receptor mutations were detected in about 53% of patients with no statistically significant difference to patient's overall survival. Anaplastic lymphoma kinase rearrangement was found in 8 out of 175 patients, harboring this abnormality leads to shorter overall survival. No correlation has been found between SLC34A2 expression and epidermal growth factor receptor mutation or anaplastic lymphoma kinase rearrangements in lung adenocarcinoma. High expression of SLC34A2 is present in about 3/4 lung adenocarcinoma samples and predicts better outcome. Since it is a membrane protein, antibody-based drugs targeting this marker might bring new resolution to this deadly disease.

Enhanced SLC34A2 in breast cancer stem cell-like cells induces chemotherapeutic resistance to doxorubicin via SLC34A2-Bmi1-ABCC5 signaling.

Even though early detection methods and treatment options are greatly improved, chemoresistance is still a tremendous challenge for breast cancer therapy. Breast cancer stem cells (BCSCs) represent a subpopulation that is central to chemoresistance. We aim to investigate the relationship between SLC34A2 and chemoresistance in BCSCs and identify the underlying mechanisms by which SLC34A2 regulates chemoresistance in BCSCs. Fluorescence Activated Cell Sorting (FACS) analysis showed the presence of a variable fraction of CD44(+)CD24(-) cells in 25 out of 25 breast cancer samples. We cultured primary breast cancer sample cells and breast cancer cell line cells to induce sphere formation in serum-free medium. Following sorting of CD44(+)CD24(-) cells from spheres, we showed that CD44(+)CD24(-) cells displayed stem cell-like features and were resistant to chemotherapy drug doxorubicin. Significantly, enhanced SLC34A2 expression correlated with chemoresponse and survival of breast cancer patients. We subsequently indicated that increased SLC34A2 expression in BCSCs directly contributed to their chemoresistance by a series of in vitro and in vivo experiments. Furthermore, we demonstrated that SLC34A2 induced chemoresistance in BCSCs via SLC34A2-Bmi1-ABCC5 signaling. Finally, we showed that ABCC5 was a direct transcriptional target of Bmi1 by chromatin immunoprecipitation (ChIP). In conclusion, our work indicated that decreased SLC34A2 expression sensitized BCSCs to doxorubicin via SLC34A2-Bmi1-ABCC5 signaling and shed new light on understanding the mechanism of chemoresistance in BCSCs. This study not only bridges the missing link between stem cell-related transcription factor (Bmi1) and ABC transporter (ABCC5) but also contributes to development of potential therapeutics against breast cancer.

Up-Regulation of Intestinal Phosphate Transporter NaPi-IIb (SLC34A2) by the Kinases SPAK and OSR1.

BACKGROUND/AIMS: SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1), kinases controlled by WNK (with-no-K[Lys] kinase), are powerful regulators of cellular ion transport and blood pressure. Observations in gene-targeted mice disclosed an impact of SPAK/OSR1 on phosphate metabolism. The present study thus tested whether SPAK and/or OSR1 contributes to the regulation of the intestinal Na(+)-coupled phosphate co-transporter NaPi-IIb (SLC34A2). METHODS: cRNA encoding NaPi-IIb was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type SPAK, constitutively active (T233E)SPAK, WNK insensitive (T233A)SPAK, catalytically inactive (D212A)SPAK, wild-type OSR1, constitutively active (T185E)OSR1, WNK insensitive (T185A)OSR1 or catalytically inactive (D164A)OSR1. The phosphate (1 mM)-induced inward current (I(Pi)) was taken as measure of phosphate transport. RESULTS: I(Pi) was observed in NaPi-IIb expressing oocytes but not in water injected oocytes, and was significantly increased by co-expression of SPAK, (T233E)SPAK, OSR1, (T185E)OSR1 or SPAK+OSR1, but not by co-expression of (T233A)SPAK, (D212A)SPAK, (T185A)OSR1, or (D164A)OSR1. SPAK and OSR1 both increased the maximal transport rate of the carrier. CONCLUSIONS: SPAK and OSR1 are powerful stimulators of the intestinal Na+-coupled phosphate co-transporter NaPi-IIb.

The effects and mechanisms of SLC34A2 in tumorigenesis and progression of human non-small cell lung cancer.

BACKGROUND: SLC34A2 with highest expressions in lung, small intestine and kidney encoded a type 2b sodium-dependent phosphate transporter (NaPi-IIb). In lung, SLC34A2 only expressed in the apical membrane of type II alveolar epithelium cells (ATII cells) and played a pivotal role during the fetal lung development and embryonic development. ATII cells acting as multifunctional stem cells might transform into NSCLC after undergoing exogenous or endogenous factors. Increasing evidences showed that the genes performing critical roles during embryogenesis were also expressed during the development of cancer. In addition, recent research found the expression of SLC34A2 had a significant difference between the surgical samples of NSCLC and normal tissues, and SLC34A2 was down-regulated in lung adenocarcinoma cell line A549 and up-regulation expression of SLC34A2 could significantly inhibit cell viability and invasion of A549 in vitro. These results suggested SLC34A2 might play an important role in the development of NSCLC. However, the role of SLC34A2 in tumorigenesis and progression of NSCLC remains unknown. RESULTS: Our study found that SLC34A2 was also significantly down-regulated in 14/15 of examined NSCLC tissues. Moreover, we found that expressions of SLC34A2 were reduced in six NSCLC cell lines for the first time. Our result also revealed a dramatic inhibitory effects of SLC34A2 on cell growth, migration and invasion of several NSCLC cell lines. SLC34A2 also strongly inhibited tumor growth and metastasis ability in A549 subcutaneous tumor model and lung metastasis model, respectively. Further studies found that the suppressive effects of SLC34A2 on tumorigenesis and progression might be associated with the down-regulation of related protein in PI3K/Akt and Ras/Raf/MEK signal pathway. CONCLUSIONS: For the first time, our data indicated that SLC34A2 could exert significantly suppressive effects on tumorigenesis and progression of NSCLC. SLC34A2 might provide new insights for further understanding the early pathogenesis of human NSCLC.

NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.

P(i) uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P(i) but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2(BBE) cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1(-/-) mice, but not PDZK1(-/-) mice, had a diminished adaptation of NaPi-2b expression in response to a low P(i) diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.

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.

Myosin VI is required for maintenance of brush border structure, composition, and membrane trafficking functions in the intestinal epithelial cell.

Characterization of the intestinal epithelium of the Snell's waltzer (sv/sv) mouse revealed that myosin VI (Myo6) is required for proper brush border (BB) ultrastructure, composition and membrane traffic. The defects observed were distinct from that observed in the myosin Ia KO, even though Myo6 is lost from the BB in this KO. Myo6 is expressed throughout the length of the small and large intestine; it is localized to the subapical inter-microvillar (MV) domain and basolateral membrane. Defects in the BB include apparent lifting of the plasma membrane off of the actin cytoskeleton in the inter-MV region, fusion of MV, and disorganized morphology of the terminal web. The molecular composition of the sv/sv BB is altered. This includes increased expression of myosin Va, myosin Ie and the MV actin binding proteins espin and phosphorylated-ezrin; myosin Id is reduced. Changes in endocytic components include reduced clathrin and adaptin ß, and increased disabled-2. Endocytic uptake of lumenal lactoferrin is inhibited in adult, but not neonatal intestinal epithelial cells. There is increased BB membrane-associated expression of both the Na(+)/H(+) exchanger, NHE3 and the Na(+)/phosphate transporter, NaPi2b. These results suggest that Myo6 is involved in the regulated trafficking of NHE3 and NaPi2b between the BB membrane and endosome.

SLC34A2 as a novel marker for diagnosis and targeted therapy of breast cancer.

The purpose of this study was to estimate the role of the SLC34A2 gene in breast cancer. A total of 146 samples were collected from breast cancer tissues and their adjacent normal breast tissues. Reverse transcription and real-time polymerase chain reaction were used to estimate gene expression levels. There was a significantly increased gene expression of SLC34A2 (normal tissues: 6.71±0.77; tumour tissues: 10.29±0.80) among breast cancer tissues compared with normal tissues. However, there was no significant association between overall survival and the gene expression level of SLC34A2. Moreover, a significant overexpression of CA125 (normal tissues: 7.26±0.62; tumour tissues: 10.51±0.58) in breast cancer tissues and a significant correlation between SLC34A2 and CA125 gene expressions were found. Our results suggested SLC34A2 to be involved in the development of breast cancer; this gene may therefore be a novel marker for the detection of breast cancer and act as a target gene in therapeutic strategies.

Nuclear xenobiotic receptor PXR-null mouse exhibits hypophosphatemia and represses the Na/Pi-cotransporter SLC34A2.

OBJECTIVE: We previously found that the lack of nuclear xenobiotic receptor, PXR, decreases femoral bone mineral density (BMD) in Pxr-/- mice. Our present study aims to elucidate the inherited phenotype that correlates with the decreased BMD and to identify the PXR-regulated gene that may link with this phenotype. METHODS: Pxr+/+ and Pxr-/- mice were used to measure the serum levels of inorganic phosphate (Pi), calcium and vitamin D3. Real time PCR and western blots were used to determine the intestinal and renal expressions of Pi and calcium transporters and various other genes involved in bone homeostasis. Cell-based reporter and gel shift assays were performed to characterize the promoter of the identified PXR-regulated gene. RESULTS: In both Pxr-/- male and female mice, lumbar, sternum, and skull were all also found to have decreased their BMD values. Serum Pi levels, but not calcium levels, are attenuated in Pxr-/- mice, exhibiting a phenotype of hypophosphatemia. Among the members of the Na/Pi contransporter family, only the SLC34A2 mRNA and protein are repressed in Pxr-/- mice. PXR can directly activate the transcription of the SLC34A2 gene through an ER6 motif on its promoter. CONCLUSION: Pxr-/- mice show the inherited phenotype of hypophosphatemia. The lack of PXR results in a severe repression of the Na/Pi cotransporter NaPi-IIb/Npt2b (SLC34A2), thus leading Pxr-/- males and females to develop a type of hypophosphatemia.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate.

Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P(i) absorption is one of the pharmacological targets that prevents it. The type II Na-P(i) cotransporter NaPi-2b is the major transporter that mediates P(i) reabsorption in the intestine. The potential role and regulation of other Na-P(i) transporters remain unknown. We have identified expression of the type III Na-P(i) cotransporter PiT-1 in the apical membrane of enterocytes. Na-P(i) transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P(i) diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P(i) transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P(i) diet, there is an increase in BBM Na-P(i) transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P(i) that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P(i) transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.

Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas.

Mouse monoclonal antibody MX35 was developed against ovarian cancer. The antibody showed homogeneous reactivity with approximately 90% of human ovarian epithelial cancers and with a limited number of normal tissues by immunohistochemistry. Although mAb MX35 has been used in a number of clinical trials in ovarian cancer, it has been difficult to define the molecular identity of MX35. We report here that mAb MX35 recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b) in human cancer cells. This conclusion is based on several lines of experimental evidence, including 1) the identification of SLC34A2, the gene coding for NaPi2b, by immunoscreening an ovarian cancer cell line cDNA expression library with mAb MX35; 2) mass spectrometry sequencing of peptides obtained by fragmentation from mAb MX35 affinity-purified antigen, which show complete sequence homology to amino acid sequences in NaPi2b; 3) selective down-regulation of SLC34A2 gene expression by RNA interference and the resulting loss of mAb MX35 binding to MX35-expressing human cancer cells; and 4) the demonstration of specific mAb MX35 reactivity with recombinant fusion proteins and with synthetic peptides of the putative largest extracellular loop of NaPi2b. We further show that NaPi2b in cancer cells is expressed on the cell surface as a heavily N-glycosylated protein, with evidence of additional post-translational modifications such as palmitoylation and the formation of disulfide bridges in the major extracellular loop. Membrane transporter molecules, such as NaPi2b, represent a new family of potential cell surface targets for the immunotherapy of cancer with monoclonal antibodies.

Chronic candesartan alters expression and activity of NKCC2, NCC, and ENaC in the obese Zucker rat.

The obese Zucker rat reportedly has increased activity of the intrarenal renin-angiotensin-aldosterone system, which conceptually could contribute to elevated salt sensitivity and blood pressure (BP). Our aim was to determine whether there was increased angiotensin II type 1 receptor (AT(1)R)-mediated upregulation of expression or activity of the bumetanide-sensitive Na-K-2Cl cotransporter, the thiazide-sensitive Na-Cl cotransporter (NCC), and/or the epithelial sodium channel (ENaC) in obese vs. lean Zucker rats. Male obese and lean Zucker rats (10-wk old) were fed either 1) control chow (1% NaCl) or 2) chow with candesartan (CAN), an AT(1)R antagonist (25 mg/kg.diet) for 14 wk (n = 8/treatment/body type). BP measured by radiotelemetry, was markedly reduced by CAN ( approximately 20-25 mmHg) in both lean and obese rats with no body-type differences. Obese rats had significantly greater net natriuretic response to single injections of hydrochlorothiazide and benzamil, suggesting increased activity of NCC and ENaC, respectively; however, only the response to benzamil was reduced by CAN. CAN led to a significant reduction in whole kidney levels of NCC and gamma-ENaC (70-kDa band) in both lean and obese rats. However, it significantly increased alpha-ENaC and Na-K-2Cl cotransporter levels, and these increases were greater in obese rats. These studies suggest that relatively increased ENaC, but not NCC activity, in obese rats is due to enhanced AT(1)R activity. CAN attenuated the reduction of several renal transporters in the obese rat kidney. Finally, differences in intrarenal AT(1)R activity do not seem directly responsible for BP differences between lean and obese rats.

Phex mutation causes the reduction of npt2b mRNA in teeth.

Hyp mice (murine homologue of human X-linked hypophosphatemia) have a disorder in phosphate homeostasis, and display hypomineralization in bones and teeth. We investigated whether a mutation of Phex (phosphate regulating gene homologies to endopeptidase on the X chromosome) has an effect on the expression level of type II sodium-dependent phosphate co-transporter (Npt2) in the developing teeth of the Hyp mouse. Quantitative RT-PCR analyses revealed that the amount of Npt2b mRNA, an isoform of Npt2, in Hyp mouse tooth germs was significantly lower than that in wild-type mice, in both in vivo and in vitro experiments. In addition, tooth germs from wild-type mice cultured in medium supplemented with antisense oligo-deoxynucleotide for Phex also showed a reduction of Npt2b mRNA expression. These findings suggest that the loss of Phex function is related to the defect of Npt2b expression in teeth, and Npt2b reduction is an intrinsic defect of Hyp murine teeth.