SLC34A2 promotes cell proliferation by activating STX17-mediated autophagy in esophageal squamous cell carcinoma.

BACKGROUND: Solute carrier family 34 member 2 (SLC34A2) has been implicated in the development of various malignancies. However, the clinical significance and underlying molecular mechanisms of SLC34A2 in esophageal squamous cell carcinoma (ESCC) remain elusive. METHODS: Western blotting, quantitative real-time PCR and immunohistochemistry were utilized to evaluate the expression levels of SLC34A2 mRNA/protein in ESCC cell lines or tissues. Kaplan-Meier curves were employed for survival analysis. CCK-8, colony formation, EdU and xenograft tumor model assays were conducted to determine the impact of SLC34A2 on ESCC cell proliferation. Cell cycle was examined using flow cytometry. RNA-sequencing and enrichment analysis were carried out to explore the potential signaling pathways. The autophagic flux was evaluated by western blotting, mRFP-GFP-LC3 reporter system and transmission electron microscopy. Immunoprecipitation and mass spectrometry were utilized for identification of potential SLC34A2-interacting proteins. Cycloheximide (CHX) chase and ubiquitination assays were conducted to test the protein stability. RESULTS: The expression of SLC34A2 was significantly upregulated in ESCC and correlated with unfavorable clinicopathologic characteristics particularly the Ki-67 labeling index and poor prognosis of ESCC patients. Overexpression of SLC34A2 promoted ESCC cell proliferation, while silencing SLC34A2 had the opposite effect. Moreover, SLC34A2 induced autophagy to promote ESCC cell proliferation, whereas inhibition of autophagy suppressed the proliferation of ESCC cells. Further studies showed that SLC34A2 interacted with an autophagy-related protein STX17 to promote autophagy and proliferation of ESCC cells by inhibiting the ubiquitination and degradation of STX17. CONCLUSIONS: These findings indicate that SLC34A2 may serve as a prognostic biomarker for ESCC.

Empagliflozin drives ferroptosis in anoikis-resistant cells by activating miR-128-3p dependent pathway and inhibiting CD98hc in breast cancer.

A tumour suppressor miRNA, miR-128-3p, is widely involved in various biological processes and has been found to get downregulated in breast cancer patients. We previously published that ectopically expressed miR-128-3p suppressed migration, invasion, cell cycle arrest, and breast cancer stem cells. In the present study, we explored the role of Empagliflozin (EMPA) as a miR-128-3p functionality-mimicking drug in inducing ferroptosis by inhibiting CD98hc. Given that CD98hc is one of the proteins critical in triggering ferroptosis, we confirmed that miR-128-3p and EMPA inhibited SP1, leading to inhibition of CD98hc expression. Further, transfection with siCD98hc, miR-128-3p mimics, and inhibitors was performed to assess their involvement in the ferroptosis of anoikis-resistant cells. We proved that anoikis-resistant cells possess high ROS and iron levels. Further, miR-128-3p and EMPA treatments induced ferroptosis by inhibiting GSH and enzymatic activity of GPX4 and also induced lipid peroxidation. Moreover, EMPA suppressed bioluminescence of 4T1-Red-FLuc induced thoracic cavity, peritoneal tumour burden and lung nodules in an in-vivo metastatic model of breast cancer. Collectively, we revealed that EMPA sensitized the ECM detached cells to ferroptosis by synergically activating miR-128-3p and lowering the levels of SP1 and CD98hc, making it a potential adjunct drug for breast cancer chemotherapy.

Severe pulmonary hypertension in pulmonary alveolar microlithiasis: A comprehensive literature review.

This review focuses on Pulmonary Alveolar Microlithiasis (PAM), an autosomal recessive genetic disorder characterized by calcium crystal deposits (microliths) resulting from loss of function of the SLC34A2 gene. PAM is a rare disease with approximately 1100 reported cases globally. The historical context of its discovery and the genetic, epidemiological, and pathophysiological aspects are discussed. PAM falls under interstitial lung diseases and is associated with pulmonary hypertension (PH), primarily categorized as Group 3 PH. The clinical manifestations, diagnostic approaches, and challenging aspects of treatment are explored. A clinical case of PAM with severe pulmonary hypertension is presented, emphasizing the importance of comprehensive evaluation and the potential benefits of phosphodiesterase-5 inhibitors (PDE5i) therapy. Despite limited therapeutic options and challenging diagnosis, this review sheds light on recent developments and emerging treatments for PAM and associated pulmonary hypertension.

CD74/SLC34A2-ROS1 Fusion Variants Involving the Transmembrane Region Predict Poor Response to Crizotinib in NSCLC Independent of TP53 Mutations.

INTRODUCTION: Variable partners and breakpoints have been reported in patients with ROS1-rearranged NSCLC. Here, we investigated the association of fusion partners and breakpoints with crizotinib efficacy in NSCLCs with common ROS1 fusions. METHODS: DNA and RNA next-generation sequencing (NGS) and immunohistochemistry were performed to characterize ROS1 fusions. RESULTS: Using DNA NGS, we identified ROS1 fusions in 210 cases, comprising 171 common (CD74/EZR/TPM3/SDC4/SLC34A2-ROS1) and 39 uncommon (variants identified in <5%) ROS1 fusion cases. DNA NGS detected variable ROS1 genomic breakpoints in common ROS1 fusions, whereas RNA NGS found ROS1 breakpoints mainly occurring in exons 32, 34 and 35, resulting in long (exon 32) and short (exon 34 or 35) ROS1 fusions. ROS1 immunohistochemistry revealed that membranous and cytoplasmic staining was predominant in long ROS1 fusions, whereas cytoplasmic staining was predominant in short ROS1 fusions (p = 0.006). For patients who received first-line crizotinib, median progression-free survival (mPFS) was lower in patients with long ROS1 fusions than those with short ROS1 fusions (8.0 versus 24.0 mo, p = 0.006). Moreover, mPFS for patients with and without TP53 mutations was 8.0 and 19.0 months, respectively (p = 0.159); mPFS for patients with and without BIM deletion polymorphism was 5.0 and 22.0 months, respectively (p = 0.003). When analyzing together with fusion partners, patients with long CD74/SLC34A2-ROS1 fusions were found to have shorter PFS than those with other ROS1, regardless of the presence or absence of TP53 mutations (p < 0.001 and p = 0.002, respectively). CONCLUSIONS: Long CD74/SLC34A2-ROS1 fusions, which retain transmembrane regions in ROS1 and fusion partners, are associated with poor response to crizotinib independent of TP53 mutations.

Alectinib relieves ischemic strokes caused by left atrial metastasis in a NSCLC patient with a novel SLC34A2-ALK (exon 1: exon 15) fusion.

BACKGROUND: The adoption of molecular profiling in non-small cell lung cancers (NSCLC) have promoted the discoveries of novel anaplastic lymphoma kinase (ALK) mutation patterns including rare intergenic rearrangements. It is always meaningful to report the structure of these fusions and their responses to ALK-inhibitors for future reference. Reports of cerebral ischemic strokes caused by atrial metastases through lymphohematogeneous spread are scarce. CASE PRESENTATION: A 35-year-old woman with no history of astherosclerosis presented with sudden onset of diplopia and facial palsy. Brain MRI scan discovered multiple infarcts around cortical and subcortical areas supplied by bilateral middle cerebral arteries, the occlusions of which were confirmed by angiography. Echocardiogram revealed intracavity appendages in atriums. The histology following valve debridement displayed endocardial metastases from lung cancer on mitral and trucuspid valves. PET/CT found right lower lobe primary tumor and mediastinal lymphadenopathies. The histology of primary lung tumor suggested adenocarcinoma and a DNA-based next-generation sequencing (NGS) test uncovered an intergenic (FAM49A, RAD51AP2)-ALK (intergenic: A14) rearrangement. Further RNA-based NGS uncovered a novel SLC34A2-ALK (exon 1: exon 15) fusion. Strokes recurred after valve surgery and vegetations reappeared on the mitral valve. Alectinib 600 mg bid was administered based on molecular finding and achieved remarkable tumor regression. Neurologic symptoms were largely relieved. No new infarctions or cerebral metastases has ever been found since. CONCLUSIONS: We report a novel SLC34A2-ALK rearrangement responding well to alectinib in a very interesting case of peripheral lung adenocarcinoma presenting with recurrent cerebral ischemic strokes due to endocardial metastases.

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.

Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis.

BACKGROUND: Variants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available. So far, 30 allelic variants in patients have been reported but only a few have been functionally characterised. This study aimed to determine the impact of selected SLC34A2 variants on transporter expression and phosphate uptake in cellular studies. METHODS: Two nonsense variants (c.910A > T and c.1456C > T), one frameshift (c.1328delT), and one in-frame deletion (c.1402_1404delACC) previously reported in patients with PAM were selected for investigation. Wild-type and mutant c-Myc-tagged human NaPi-IIb constructs were expressed in Xenopus laevis oocytes. The transport function was investigated with a 32Pi uptake assay. NaPi-IIb protein expression and localisation were determined with immunoblotting and immunohistochemistry, respectively. RESULTS: Oocytes injected with the wild-type human NaPi-IIb construct had significant 32Pi transport compared to water-injected oocytes. In addition, the protein had a molecular weight as expected for the glycosylated form, and it was readily detectable in the oocyte membrane. Although the protein from the Thr468del construct was synthesised and expressed in the oocyte membrane, phosphate transport was similar to non-injected control oocytes. All other mutants were non-functional and not expressed in the membrane, consistent with the expected impact of the truncations caused by premature stop codons. CONCLUSIONS: Of four analysed SLC34A2 variants, only the Thr468del showed similar protein expression as the wild-type cotransporter in the oocyte membrane. All mutant transporters were non-functional, supporting that dysfunction of NaPi-IIb underlies the pathology of PAM.

Pulmonary Alveolar Microlithiasis: A novel patient and brief review of the literature.

Pulmonary Alveolar Microlithiasis (PAM) is a rare hereditary lung disease caused by biallelic pathogenic variants (pV) in the solute family 34 member 2 gene (SLC34A2; Izumi et al., Am J Respir Crit Care Med 2007; 175: 263-268). pVs in this sodium phosphate co-transporter gene lead to accumulation of calcium phosphate crystals within pulmonary alveoli. More than 1000 cases of PAM were thus far reported, with high variance in disease courses (Stamatis et al., Ann Thorac Surg 1993; 56: 972-975). Frequently, asymptomatic cases are observed, and often times slow disease progression until respiratory insufficiency in middle age occurs (Kosciuk, Eur Respir Rev 2020; 29: 200024). Treatment options for PAM are scarce and largely ineffective, and lung transplantation is the only effective therapy in end-stage disease (Stamatis et al., Ann Thorac Surg 1993; 56: 972-975). Here, we report a novel PAM case in an adolescent migrant from East Africa and discuss current diagnostic and therapeutic options.

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.

Discovery of Gut-Restricted Small-Molecule Inhibitors of Intestinal Sodium-Dependent Phosphate Transport Protein 2b (NaPi2b) for the Treatment of Hyperphosphatemia.

NaPi2b is primarily expressed in the small intestine, lungs, and testes and plays an important role in phosphate homeostasis. The inhibition of NaPi2b, responsible for intestinal phosphate absorption, is considered to reduce serum phosphate levels, making it a promising therapeutic approach for hyperphosphatemia. Using a novel phosphate uptake inhibitor 3 (IC50 = 87 nM), identified from an in-house compound collection in human NaPi2b-transfected cells as a prototype compound, we conducted its derivatization based on a Ro5-deviated strategy to develop orally administrable small-molecule NaPi2b inhibitors with nonsystemic exposure. Consequently, compound 15, a zwitterionic compound with a potent in vitro phosphate uptake inhibitory activity (IC50 = 64 nM) and a low membrane permeability (Pe < 0.025 × 10-6 cm/s), was developed. Compound 15 showed a low bioavailability (F = 0.1%) in rats and a reduction in phosphate absorption in the rat intestinal loop assay comparable to sevelamer hydrochloride, a clinically effective phosphate binder for treating hyperphosphatemia.

SLC34A2 promotes cancer proliferation and cell cycle progression by targeting TMPRSS3 in colorectal cancer.

Solute carrier family 34 member 2 (SLC34A2), a family member of sodium-driven phosphate cotransporters, has been reported to facilitate cell proliferation and tumor growth. However, the functional mechanism by which SLC34A2 promotes cell growth and cell cycle progression remains poorly understood. Here, we reported that SLC34A2 was overexpressed in CRC by analysis of TCGA and GEO datasets. A total of 45 differentially expressed genes (DEGs) were identified from comparing SLC34A2-high or -low groups and functional enrichment analysis of these DEGs demonstrated that cell cycle pathway was enriched. Interestingly, we found a positive correlation between TMPRSS3 (transmembrane serine protease 3) and SLC34A2, which was confirmed by RT-qPCR and western blotting. Furthermore, TMPRSS3 was also upregulated in CRC tumor tissues compared to normal tissues. Patients with high TMPRSS3 expression had poor prognosis. Functionally, TMPRSS3 deficiency inhibited cell proliferation and colony formation in CRC cells. TMPRSS3 overexpression in SLC34A2-deficient cells antagonized siSLC34A2-mediated cell cycle inhibition by promoting cyclin E, cyclin A protein expression. Based on these results, our study suggests that SLC34A2 promotes cancer proliferation and cell cycle progression by targeting TMPRSS3 in colorectal cancer cells.

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.

Pulmonary Alveolar Microlithiasis - A Review.

Pulmonary Alveolar Microlithiasis (PAM) is a rare genetic disorder causing widespread deposition of calcium-phosphate crystals in the alveolar space. A hallmark of the disease is the discrepancy between perceived symptoms upon diagnosis compared with the extensive, sandstorm-like appearance of the microliths on chest X-ray or HRCT. Caused by a defective sodium-dependent phosphate transport protein due to loss-of-function variants of the SLC34A2 gene, PAM is an autosomal recessive transmitted disorder, and as such has a high correlation to consanguinity. The most common variants of the SLC34A2 gene are single nucleotide biallelic changes, but larger deletions are described. Initial suspicion of PAM on radiological examination should be followed by genetic testing to verify the diagnosis and identify the disease-causing variant. When not available, the diagnosis can be made by means of invasive techniques, such as transbronchial forceps or cryobiopsy, or a surgical lung biopsy. In families with a history of PAM, genetic counseling should be offered, as well as preimplantation/prenatal testing if necessary. As of writing this review, no definitive treatment exists, and PAM may in some cases progress to severe pulmonary disease with respiratory failure and potential death. Patients with PAM should be offered preventative and symptomatic treatments such as vaccinations and oxygen therapy when needed. In some cases, lung transplantation may be required.