New insights in the genetic variant spectrum of SLC34A2 in pulmonary alveolar microlithiasis; a systematic review.

Pulmonary alveolar microlithiasis (PAM) is a rare autosomal recessive lung disease caused by variants in the SLC34A2 gene encoding the sodium-dependent phosphate transport protein 2B, NaPi-2b. PAM is characterized by deposition of calcium phosphate crystals in the alveoli. Onset and clinical course vary considerably; some patients remain asymptomatic while others develop severe respiratory failure with a significant symptom burden and compromised survival. It is likely that PAM is under-reported due to lack of recognition, misdiagnosis, and mild clinical presentation. Most patients are genetically uncharacterized as the diagnostic confirmation of PAM has traditionally not included a genetic analysis. Genetic testing may in the future be the preferred tool for diagnostics instead of invasive methods. This systematic review aims to provide an overview of the growing knowledge of PAM genetics. Rare variants in SLC34A2 are found in almost all genetically tested patients. So far, 34 allelic variants have been identified in at least 68 patients. A majority of these are present in the homozygous state; however, a few are found in the compound heterozygous form. Most of the allelic variants involve only a single nucleotide. Half of the variants are either nonsense or frameshifts, resulting in premature termination of the protein or decay of the mRNA. There is currently no cure for PAM, and the only effective treatment is lung transplantation. Management is mainly symptomatic, but an improved understanding of the underlying pathophysiology will hopefully result in development of targeted treatment options. More standardized data on PAM patients, including a genetic diagnosis covering larger international populations, would support the design and implementation of clinical studies to the benefit of patients. Further genetic characterization and understanding of how the molecular changes influence disease phenotype will hopefully allow earlier diagnosis and treatment of the disease in the future.

Transient DUX4 expression in human embryonic stem cells induces blastomere-like expression program that is marked by SLC34A2.

Embryonic genome activation (EGA) is critical for embryonic development. However, our understanding of the regulatory mechanisms of human EGA is still incomplete. Human embryonic stem cells (hESCs) are an established model for studying developmental processes, but they resemble epiblast and are sub-optimal for modeling EGA. DUX4 regulates human EGA by inducing cleavage-stage-specific genes, while it also induces cell death. We report here that a short-pulsed expression of DUX4 in primed hESCs activates an EGA-like gene expression program in up to 17% of the cells, retaining cell viability. These DUX4-induced cells resembled eight-cell stage blastomeres and were named induced blastomere-like (iBM) cells. The iBM cells showed marked reduction of POU5F1 protein, as previously observed in mouse two-cell-like cells. Finally, the iBM cells were successfully enriched using an antibody against NaPi2b (SLC34A2), which is expressed in human blastomeres. The iBM cells provide an improved model system to study human EGA transcriptome.

Effects of pharmacological inhibition of the sodium-dependent phosphate cotransporter 2b (NPT2b) on intestinal phosphate absorption in mouse and rat models.

An excess phosphate burden in renal disease has pathological consequences for bone, kidney, and heart. Therapies to decrease intestinal phosphate absorption have been used to address the problem, but with limited success. Here, we describe the in vivo effects of a novel potent inhibitor of the intestinal sodium-dependent phosphate cotransporter NPT2b, LY3358966. Following treatment with LY3358966, phosphate uptake into plasma 15 min following an oral dose of radiolabeled phosphate was decreased 74% and 22% in mice and rats, respectively, indicating NPT2b plays a much more dominant role in mice than rats. Following the treatment with LY3358966 and radiolabeled phosphate, mouse feces were collected for 48 h to determine the ability of LY3358966 to inhibit phosphate absorption. Compared to vehicle-treated animals, there was a significant increase in radiolabeled phosphate recovered in feces (8.6% of the dose, p < .0001). Similar studies performed in rats also increased phosphate recovered in feces (5.3% of the dose, p < .05). When used in combination with the phosphate binder sevelamer in rats, there was a further small, but not significant, increase in fecal phosphate. In conclusion, LY3358966 revealed a more prominent role for NPT2b on acute intestinal phosphate uptake into plasma in mice than rats. However, the modest effects on total intestinal phosphate absorption observed in mice and rats with LY3359866 when used alone or in combination with sevelamer highlights the challenge to identify new more effective therapeutic targets and/or drug combinations to treat the phosphate burden in patients with renal disease.

Design, synthesis and biological evaluation of novel 1H-pyrazole-4-carbonyl-4,5,6,7-tetrahydrobenzo [b]thiophene derivatives as gut-selective NaPi2b inhibitors.

Intestinal sodium-dependent phosphate transport protein 2b (SLC34A2, NaPi2b) inhibitors are expected to be potential new candidates for anti-hyperphosphatemia drugs. However, a risk of on-target side effects based on the inhibition of NaPi2b in the lung and testis has been reported. To identify gut-selective (minimally systemic) NaPi2b inhibitors, we prepared and evaluated 1H-pyrazole-4-carbonyl-4,5,6,7-tetrahydrobenzo[b]thiophene derivatives with highly polar functional groups to reduce systemic exposure. As a result, compounds 36a and 36b showed a good activity in vitro and a low bioavailability in Sprague-Dawley (SD) rats. However, these compounds did not suppress phosphate absorption in SD rats. This lack of in vivo efficacy could be due to the high hydrophobicity of these compounds. The results of further investigations of other classes of compounds with appropriate physical properties will be reported in due course.

NaPi2b expression in a large surgical Non-Small Cell Lung Cancer (NSCLC) cohort.

BACKGROUND: NaPi2b is a multi-transmembrane sodium-dependent phosphate transporter expressed at normal levels in several organs, including lung. High expression levels have been reported in various tumors including breast, thyroid, ovarian and non-small cell lung cancer. To date evaluation of NaPi2b expression has mostly been restricted to smaller lung cancer cohorts. METHODS: Analyses were performed on archival formalin fixed paraffin embedded primary tumor specimens from patients who had undergone curative intent resection at an Australian tertiary hospital. Tissue microarrays were constructed and stained with the chimeric anti-NaPi2b antibody, MERS67. Semi-quantitative H-scores (range 0 - 300) were calculated for each core tissue sample (H-score = % tumor cells staining for NaPi2b multiplied by staining intensity). An overall average H-score was reported for each specimen, with a cut-off score of 50 considered positive. RESULTS: Of 438 cases, high NaPi2b expression was observed in 151 (34.5%) overall, high expression in 137 of 208 (65.9%) adenocarcinoma cases, and 5 of 179 (2.8%) squamous cases (P < .0001). High NaPi2b expression was associated with female sex, EGFR or KRAS mutation, and TTF1 positivity (adenocarcinoma cases only). High NaPi2b expression was associated with improved overall survival (median 54 vs. 35 months, P = .029). CONCLUSION: High NaPi2b expression was noted in a significant subset of adenocarcinoma cases, and in particular amongst those who were TTF1+, or exhibited EGFR or KRAS mutations. This agrees with earlier reports and highlights the significance that NaPi2b may have a role as a possible target for delivery of cytotoxic agents via antibody-drug conjugate models for some patients with lung adenocarcinoma.

Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors.

BACKGROUND: The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors. METHODS: With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature. RESULTS: Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl- currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl- currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate. CONCLUSIONS: Extracellular phosphate modulates the magnitude of CFTR-mediated Cl- currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors.

Constitutive depletion of Slc34a2/NaPi-IIb in rats causes perinatal mortality.

Absorption of dietary phosphate (Pi) across intestinal epithelia is a regulated process mediated by transcellular and paracellular pathways. Although hyperphosphatemia is a risk factor for the development of cardiovascular disease, the amount of ingested Pi in a typical Western diet is above physiological needs. While blocking intestinal absorption has been suggested as a therapeutic approach to prevent hyperphosphatemia, a complete picture regarding the identity and regulation of the mechanism(s) responsible for intestinal absorption of Pi is missing. The Na+/Pi cotransporter NaPi-IIb is a secondary active transporter encoded by the Slc34a2 gene. This transporter has a wide tissue distribution and within the intestinal tract is located at the apical membrane of epithelial cells. Based on mouse models deficient in NaPi-IIb, this cotransporter is assumed to mediate the bulk of active intestinal absorption of Pi. However, whether or not this is also applicable to humans is unknown, since human patients with inactivating mutations in SLC34A2 have not been reported to suffer from Pi depletion. Thus, mice may not be the most appropriate experimental model for the translation of intestinal Pi handling to humans. Here, we describe the generation of a rat model with Crispr/Cas-driven constitutive depletion of Slc34a2. Slc34a2 heterozygous rats were indistinguishable from wild type animals under standard dietary conditions as well as upon 3 days feeding on low Pi. However, unlike in humans, homozygosity resulted in perinatal lethality.

Evidence of an intestinal phosphate transporter alternative to type IIb sodium-dependent phosphate transporter in rats with chronic kidney disease.

BACKGROUND: Phosphate is absorbed in the small intestine via passive flow and active transport.NaPi-IIb, a type II sodium-dependent phosphate transporter, is considered to mediate active phosphate transport in rodents. To study the regulation of intestinal phosphate transport in chronic kidney disease (CKD), we analyzed the expression levels of NaPi-IIb, pituitary-specific transcription factor 1 (PiT-1) and PiT-2 and the kinetics of intestinal phosphate transport using two CKD models. METHODS: CKD was induced in rats via adenine orThy1 antibody injection. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the messenger RNA (mRNA) expression of NaPi-IIb, PiT-1 and PiT-2 were analyzed. The protein expression level of NaPi-IIb was measured by mass spectrometry (e.g. liquid chromatography tandem mass spectrometry). RESULTS: In normal rats, phosphate uptake into BBMV consisted of a single saturable component and its Michaelis constant (Km) was comparable to that of NaPi-IIb. The maximum velocity (Vmax) correlated with mRNA and protein levels of NaPi-IIb. In the CKD models, intestinal phosphate uptake consisted of two saturable components. The Vmax of the higher-affinity transport, which is thought to be responsible for NaPi-IIb, significantly decreased and the decrease correlated with reduced NaPi-IIb expression. The Km of the lower-affinity transport was comparable to that of PiT-1 and -2. PiT-1 mRNA expression was much higher than that of PiT-2, suggesting that PiT-1 was mostly responsible for phosphate transport. CONCLUSIONS: This study suggests that the contribution of NaPi-IIb to intestinal phosphate absorption dramatically decreases in rats with CKD and that a low-affinity alternative to NaPi-IIb, in particular PiT-1, is upregulated in a compensatory manner in CKD.

Effects of available phosphorus source and concentration on performance and expression of sodium phosphate type IIb cotransporter, vitamin D-1α-hydroxylase, and vitamin D-24-hydroxylase mRNA in broiler chicks.

This experiment was conducted to examine the effect of 2 phosphorus (P) sources on broiler performance to day 14. The P bioavailability was estimated using bird performance and tibia ash measurements, whereas P digestibility, intestinal P transporter, kidney vitamin D-1α-hydroxylase, and vitamin D-24-hydroxylase mRNA abundances were also determined. Slope regression analysis was used to determine the bioavailability of dicalcium phosphate (Dical P) and nanocalcium phosphate (Nano P) with dietary available P (AvP) set to 0.20% P (control) using AvP from the major ingredients and Dical P. The experimental treatments were achieved by supplementation with either Dical P or Nano P to generate 0.24, 0.28, 0.32, and 0.36% AvP. A total of 648-day-old unsexed broiler chicks were divided into 72 birds per treatment (8 replicate cages of 9 birds). Slope regression analysis showed positive linear relationships between BW, feed intake (FI), tibia ash weight (TAW), and tibia ash percentage (TAP) with dietary Dical P and Nano P levels. Comparisons between regression slopes for Dical P and Nano P fed birds were not significantly different for BW, feed intake, tibia ash weight, and tibia ash percentage, indicating similar P bioavailability from Dical P and Nano P. There were interactions between P source and AvP for feed efficiency (FE) and apparent ileal P digestibility (AIPD). Dicalcium phosphate had greater FE than Nano P at 0.28% AvP and greater AIPD than Nano P at 0.24% AvP. The addition of AvP from Dical P and Nano P resulted in reduced sodium phosphate cotransporter mRNA abundance in the duodenum in a dose-dependent response. In the kidney, vitamin D-1α-hydroxylase mRNA abundance was greater at 0.36% Nano P compared with control, but there was no difference with Dical P. There was no difference in vitamin D-24-hydroxylase mRNA abundance between control and supplementation with Nano P or Dical P. In conclusion, Nano P and Dical P had the same bioavailability but had different effects on gene expression.

Vitamin D3 enhanced intestinal phosphate cotransporter genes in young and growing broilers.

The influence of dietary vitamin D3 (VD3) levels on growth, bone performance, and duodenal type IIb sodium-dependent phosphate cotransporter (NaPi-IIb) genes in broiler chicken were studied. One-day-old male Ross308 broilers (n = 432) were allocated into 6 treatment groups with each group consisting of 6 cage pens. Each treatment group received diet containing different amounts of VD3 (80, 200, 500, 1,250, 3,125, or 7,813 IU per kg of diet) from a day-old to 31 D of age. Dietary available phosphorus and calcium were kept the same across all treatments in each phase. At 14 D, influence of VD3 on BW gain was found in the birds that received VD3 of 3,125 IU/kg and 200 IU/kg (P < 0.05). Toe ash and tibia ash linearly increased (P < 0.05) at 14 D with increase in dietary VD3. There was no significant influence of dietary VD3 on tibia breaking strength. In both phases, relative expression of duodenal NaPi-IIb linearly increased (P < 0.01) with increase in dietary VD3. At 14 D, highest expression of 3.2 folds was observed in birds treated with VD3 at 7,813 IU/kg of feed. At 31 D, birds that received VD3 levels of 3,125 and 7,813 IU/kg of feed showed 2.9 folds higher in NaPi-IIb expression compared with those fed lowest level of VD3 at 80 IU/kg of feed. When dietary calcium and phosphorus were maintained at the standard requirement, increase in dietary VD3 did not improve growth performance. For optimum growth and bone characteristics, dietary inclusion of VD3 at 500 IU/kg was adequate for both starter and grower broiler diets. Vitamin D3 enhanced the expression of NaPi-IIb at higher doses and thus improving the tibia ash content in high VD3 treatment groups. This study reported for the first time an increased in the expression of duodenal NaPi-IIb in 31-day-old broilers in response to high dietary VD3 levels.

Expression of NaPi-IIb in rodent and human kidney and upregulation in a model of chronic kidney disease.

Na+-coupled phosphate cotransporters from the SLC34 and SLC20 families of solute carriers mediate transepithelial transport of inorganic phosphate (Pi). NaPi-IIa/Slc34a1, NaPi-IIc/Slc34a3, and Pit-2/Slc20a2 are all expressed at the apical membrane of renal proximal tubules and therefore contribute to renal Pi reabsorption. Unlike NaPi-IIa and NaPi-IIc, which are rather kidney-specific, NaPi-IIb/Slc34a2 is expressed in several epithelial tissues, including the intestine, lung, testis, and mammary glands. Recently, the expression of NaPi-IIb was also reported in kidneys from rats fed on high Pi. Here, we systematically quantified the mRNA expression of SLC34 and SLC20 cotransporters in kidneys from mice, rats, and humans. In all three species, NaPi-IIa mRNA was by far the most abundant renal transcript. Low and comparable mRNA levels of the other four transporters, including NaPi-IIb, were detected in kidneys from rodents and humans. In mice, the renal expression of NaPi-IIa transcripts was restricted to the cortex, whereas NaPi-IIb mRNA was observed in medullary segments. Consistently, NaPi-IIb protein colocalized with uromodulin at the luminal membrane of thick ascending limbs of the loop of Henle segments. The abundance of NaPi-IIb transcripts in kidneys from mice was neither affected by dietary Pi, the absence of renal NaPi-IIc, nor the depletion of intestinal NaPi-IIb. In contrast, it was highly upregulated in a model of oxalate-induced kidney disease where all other SLC34 phosphate transporters were downregulated. Thus, NaPi-IIb may contribute to renal phosphate reabsorption, and its upregulation in kidney disease might promote hyperphosphatemia.

Significant Species Differences in Intestinal Phosphate Absorption between Dogs, Rats, and Monkeys.

A treatment for hyperphosphatemia would be expected to reduce mortality rates for CKD and dialysis patients. Although rodent studies have suggested sodium-dependent phosphate transporter type IIb (NaPi-IIb) as a potential target for hyperphosphatemia, NaPi-IIb selective inhibitors failed to achieve efficacy in human clinical trials. In this study, we analyzed phosphate metabolism in rats, dogs, and monkeys to confirm the species differences. Factors related to phosphate metabolism were measured and intestinal phosphate absorption rate was calculated from fecal excretion in each species. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the mRNA expression of NaPi-IIb, PiT-1, and PiT-2 were analyzed. In addition, alkaline phosphatase (ALP) activity was evaluated. The intestinal phosphate absorption rate, including phosphate uptake by BBMV and NaPi-IIb expression, was the highest in dogs. Notably, urinary phosphate excretion was the lowest in monkeys, and their intestinal phosphate absorption rate was by far the lowest. Dogs and rats showed positive correlations between Vmax/Km of phosphate uptake in BBMV and NaPi-IIb expression. Although phosphate uptake was observed in the BBMV of monkeys, NaPi-IIb expression was not detected and ALP activity was low. This study revealed significant species differences in intestinal phosphate absorption. NaPi-IIb contributes to intestinal phosphate uptake in rats and dogs. However, in monkeys, phosphate is poorly absorbed due to the slight degradation of organic phosphate in the intestine.

SLC34A2 simultaneously promotes papillary thyroid carcinoma growth and invasion through distinct mechanisms.

Thyroid cancer is the fastest growing cancer among all solid tumors in recent decades. Papillary thyroid carcinoma (PTC) is the most predominant type of thyroid cancer. Around 30% of PTC patients with distant metastases and local invasion receive poor prognosis. Thus, the identification of new druggable biological targets is of great importance. Accumulating evidence indicates that solute carrier family numbers have emerged as obligate effectors during the progression of multiple malignancies. Here, we uncovered the functional significance, molecular mechanisms, and clinical impact of solute carrier family 34 member A2 (SLC34A2) in PTC. SLC34A2 was markedly overexpressed in PTC tissues at both mRNA and protein levels compared with matched adjacent normal tissues due to promoter hypomethylation mediated by the DNA methyltransferase 3 beta (DNMT3B). Furthermore, a series of in vivo and in vitro gain- or loss-of-functional assays elucidated the role of SLC34A2 in boosting cell proliferation, cell cycle progression, migration, invasion, and adhesion of PTC cells. Using immunoprecipitation and mass spectrometry, we discovered that SLC34A2 bound to the actin-binding repeats domain of Cortactin (CTTN), thereby inducing the invadopodia formation of PTC cells to promote the metastasis potential of PTC cells. Besides, our mechanistic studies, as well as gene set enrichment analysis (GSEA), have pinpointed the PTEN/AKT/FOXO3a pathway as a major signaling functioning downstream of SLC34A2 regulated cell growth. Taken together, our results highlighted that SLC34A2 plays a pivotal oncogenic role during carcinogenesis and metastasis through distinct mechanisms in PTC.

All-trans retinoic acid reduces the transcriptional regulation of intestinal sodium-dependent phosphate co-transporter gene (Npt2b).

Inorganic phosphate (Pi) homeostasis is regulated by intestinal absorption via type II sodium-dependent co-transporter (Npt2b) and by renal reabsorption via Npt2a and Npt2c. Although we previously reported that vitamin A-deficient (VAD) rats had increased urine Pi excretion through the decreased renal expression of Npt2a and Npt2c, the effect of vitamin A on the intestinal Npt2b expression remains unclear. In this study, we investigated the effects of treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A, on the Pi absorption and the Npt2b expression in the intestine of VAD rats, as well as and the underlying molecular mechanisms. In VAD rats, the intestinal Pi uptake activity and the expression of Npt2b were increased, but were reduced by the administration of ATRA. The transcriptional activity of reporter plasmid containing the promoter region of the rat Npt2b gene was reduced by ATRA in NIH3T3 cells overexpressing retinoic acid receptor (RAR) and retinoid X receptor (RXR). On the other hand, CCAAT/enhancer-binding proteins (C/EBP) induced transcriptional activity of the Npt2b gene. Knockdown of the C/EBP gene and a mutation analysis of the C/EBP responsible element in the Npt2b gene promoter indicated that C/EBP plays a pivotal role in the regulation of Npt2b gene transcriptional activity by ATRA. EMSA revealed that the RAR/RXR complex inhibits binding of C/EBP to Npt2b gene promoter. Together, these results suggest that ATRA may reduce the intestinal Pi uptake by preventing C/EBP activation of the intestinal Npt2b gene.

High-fat diets provoke phosphorus absorption from the small intestine in rats.

OBJECTIVE: The ratio of dietary carbohydrate to fat may affect phosphorus metabolism because both calcium and phosphorus are regulated by similar metabolic mechanisms, and a high-fat diet (HFD) induces deleterious effects on the absorption of dietary calcium. We hypothesized that an HFD induces an increase in phosphorus absorption. The aim of this study was to evaluate the effects of differences in the quantity and quality of dietary fat on phosphorus metabolism over the short- and long-term. METHODS: Eighteen 8-wk-old Sprague-Dawley male rats were fed an isocaloric diet containing varied ratios of carbohydrates to fat energy and sources of fat (control diet, HFD, and high- saturated fat diet [HF-SFA]). At 3 d and 7 wk after the allocation and initiation of the test diets, feces and urine were collected and used for phosphorus and calcium measurement. RESULTS: The fecal phosphorous concentration (F-Pi) was lower in the HF-SFA group than in the other two groups; however, the urine phosphorus concentration (U-Pi) was significantly higher in the HF-SFA group than the other two groups when the rats were fed over the short- (P < 0.01) and long -term (P < 0.01 versus control, P < 0.05 versus HFD group). There were no significant differences in type-IIa sodium-phosphate cotransporter (NaPi-2 a) and type-IIc sodium-phosphate cotransporter (NaPi-2 c) mRNA expression, which are renal phosphate transport-related genes; however, the expression of type-IIb sodium-phosphate cotransporter (NaPi-2 b) and type-III sodium-phosphate cotransporter (Pit-1) mRNA in the duodenum was higher in the HFD and HF-SFA groups than in the control group (P < 0.05), although there were no significant differences in these in the jejunum. CONCLUSIONS: The present results indicated that an HFD, particularly HF-SFA, increases intestinal phosphate absorption compared with control.

Phase Ia Study of Anti-NaPi2b Antibody-Drug Conjugate Lifastuzumab Vedotin DNIB0600A in Patients with Non-Small Cell Lung Cancer and Platinum-Resistant Ovarian Cancer.

PURPOSE: This phase I trial assessed the safety, tolerability, and preliminary antitumor activity of lifastuzumab vedotin (LIFA), an antibody-drug conjugate of anti-NaPi2b mAb (MNIB2126A) and a potent antimitotic agent (monomethyl auristatin E). PATIENTS AND METHODS: LIFA was administered to patients with non-small cell lung cancer (NSCLC) and platinum-resistant ovarian cancer (PROC), once every 3 weeks, by intravenous infusion. The starting dose was 0.2 mg/kg in this 3+3 dose-escalation design, followed by cohort expansion at the recommended phase II dose (RP2D). RESULTS: Overall, 87 patients were treated at doses between 0.2 and 2.8 mg/kg. The MTD was not reached; 2.4 mg/kg once every 3 weeks was selected as the RP2D based on overall tolerability profile. The most common adverse events of any grade and regardless of relationship to study drug were fatigue (59%), nausea (49%), decreased appetite (37%), vomiting (32%), and peripheral sensory neuropathy (29%). Most common treatment-related grade ≥3 toxicities among patients treated at the RP2D (n = 63) were neutropenia (10%), anemia (3%), and pneumonia (3%). The pharmacokinetic profile was dose proportional. At active doses ≥1.8 mg/kg, partial responses were observed in four of 51 (8%) patients with NSCLC and 11 of 24 (46%) patients with PROC per RECIST. All RECIST responses occurred in patients with NaPi2b-high by IHC. The CA-125 biomarker assessed for patients with PROC dosed at ≥1.8 mg/kg showed 13 of 24 (54%) had responses (≥50% decline from baseline). CONCLUSIONS: LIFA exhibited dose-proportional pharmacokinetics and an acceptable safety profile, with encouraging activity in patients with PROC at the single-agent RP2D of 2.4 mg/kg.

Long non-coding RNA MEG3 regulates migration and invasion of lung cancer stem cells via miR-650/SLC34A2 axis.

Long non-coding RNA maternally expressed gene 3 (MEG3) is related to the occurrence and development of non-small cell lung cancer (NSCLC). However, the function and underlying molecular mechanisms of MEG3 in lung cancer stem cells (LCSCs) are still unclear. LCSCs were determined in lung cancer cells using fluorescence-activated cell sorting (FACS). qRT-PCR and western blot were performed to examine the expressions of MEG3, miR-650, solute carrier family 34 member 2 (SLC34A2), octamer-binding transcription factor 4 (Oct4), and CD133. Sphere assay was employed to evaluate sphere-forming ability. Cell migration and invasion were analyzed by Transwell assay. The relationships among MEG3, miR-650, and SLC34A2 were validated by luciferase reporter, RIP, and RNA pulldown assays. We found MEG3 was downregulated in LCSCs. MEG3 depletion strengthened stem cell-like characteristics and sphere-forming ability in LCCs. Upregulation of MEG3 suppressed migration and invasion in LCCs and LCSCs. miR-650 was bound to MEG3 and upregulated in LCSCs. miR-650 inhibitor alleviated si-MEG3-induced promotion of stem cell-like characteristics in lung cancer cells (LCCs) H1299. Furthermore, miR-650 mimic attenuated the MEG3 upregulation-mediated inhibition of migration and invasion. In addition, SLC34A2 was a target of miR-650 and downregulated in LCSCs. miR-650 mimic induced stem cell-like characteristics in LCCs, which was weakened by overexpression of SLC34A2. In contrast, the repression of SLC34A2 mitigated the miR-650 silencing-induced inhibition of migration and invasion in LCCs and LCSCs. Besides, MEG3 regulated SLC34A2 expression by sponging miR-650. Importantly, SLC34A2 weakened MEG3-mediated stem cell-like state and cell metastasis. Our data suggested MEG3 was involved in stem cell-like state of LCCs and curbed migration and invasion through miR-650/SLC34A2 axis in NSCLC.

Paracellular transport of phosphate along the intestine.

Inorganic phosphate (Pi) is crucial for many biological functions, such as energy metabolism, signal transduction, and pH buffering. Efficient systems must exist to ensure sufficient supply for the body of Pi from diet. Previous experiments in humans and rodents suggest that two pathways for the absorption of Pi exist, an active transcellular Pi transport and a second paracellular pathway. Whereas the identity, role, and regulation of active Pi transport have been extensively studied, much less is known about the properties of the paracellular pathway. In Ussing chamber experiments, we characterized paracellular intestinal Pi permeabilities and fluxes. Dilution potential measurements in intestinal cell culture models demonstrated that the tight junction is permeable to Pi, with monovalent Pi having a higher permeability than divalent Pi. These findings were confirmed in rat and mouse intestinal segments by use of Ussing chambers and a combination of dilution potential measurements and fluxes of radiolabeled 32Pi. Both techniques yielded very similar results, showing that paracellular Pi fluxes were bidirectional and that Pi permeability was ~50% of the permeability for Na+ or Cl-. Pi fluxes were a function of the concentration gradient and Pi species (mono- vs. divalent Pi). In mice lacking the active transcellular Pi transport component sodium-dependent Pi transporter NaPi-IIb, the paracellular pathway was not upregulated. In summary, the small and large intestines have a very high paracellular Pi permeability, which may favor monovalent Pi fluxes and allow efficient uptake of Pi even in the absence of active transcellular Pi uptake.NEW & NOTEWORTHY The paracellular permeability for phosphate is high along the entire axis of the small and large intestine. There is a slight preference for monovalent phosphate. Paracellular phosphate fluxes do not increase when transcellular phosphate transport is genetically abolished. Paracellular phosphate transport may be an important target for therapies aiming to reduce intestinal phosphate absorption.