Oxidative phosphorylation is a metabolic vulnerability of endocrine therapy and palbociclib resistant metastatic breast cancers.

Resistance to endocrine treatments and CDK4/6 inhibitors is considered a near-inevitability in most patients with estrogen receptor positive breast cancers (ER + BC). By genomic and metabolomics analyses of patients' tumours, metastasis-derived patient-derived xenografts (PDX) and isogenic cell lines we demonstrate that a fraction of metastatic ER + BC is highly reliant on oxidative phosphorylation (OXPHOS). Treatment by the OXPHOS inhibitor IACS-010759 strongly inhibits tumour growth in multiple endocrine and palbociclib resistant PDX. Mutations in the PIK3CA/AKT1 genes are significantly associated with response to IACS-010759. At the metabolic level, in vivo response to IACS-010759 is associated with decreased levels of metabolites of the glutathione, glycogen and pentose phosphate pathways in treated tumours. In vitro, endocrine and palbociclib resistant cells show increased OXPHOS dependency and increased ROS levels upon IACS-010759 treatment. Finally, in ER + BC patients, high expression of OXPHOS associated genes predict poor prognosis. In conclusion, these results identify OXPHOS as a promising target for treatment resistant ER + BC patients.

Homologous recombination deficiency derived from whole-genome sequencing predicts platinum response in triple-negative breast cancers.

The high frequency of homologous recombination deficiency (HRD) is the main rationale of testing platinum-based chemotherapy in triple-negative breast cancer (TNBC), however, the existing methods to identify HRD are controversial and there is a medical need for predictive biomarkers. We assess the in vivo response to platinum agents in 55 patient-derived xenografts (PDX) of TNBC to identify determinants of response. The HRD status, determined from whole genome sequencing, is highly predictive of platinum response. BRCA1 promoter methylation is not associated with response, in part due to residual BRCA1 gene expression and homologous recombination proficiency in different tumours showing mono-allelic methylation. Finally, in 2 cisplatin sensitive tumours we identify mutations in XRCC3 and ORC1 genes that are functionally validated in vitro. In conclusion, our results demonstrate that the genomic HRD is predictive of platinum response in a large cohort of TNBC PDX and identify alterations in XRCC3 and ORC1 genes driving cisplatin response.

HORMAD1 overexpression predicts response to anthracycline-cyclophosphamide and survival in triple-negative breast cancers.

Triple negative breast cancers (TNBCs) represent 15-20% of all breast cancers and are associated with higher recurrence and distant metastasis rate. Standard of care for early stage TNBC is anthracyclines combined with cyclophosphamide (AC) followed by taxanes, in the neo-adjuvant or adjuvant setting. This work aimed to identify predictive biomarkers of AC response in patient-derived xenograft (PDX) models of TNBC and to validate them in the clinical setting. By gene and protein expression analysis of 39 PDX with different responses to AC, we found that high expression of HORMAD1 was associated with better response to AC. Both gene and protein expression were associated with promoter hypomethylation. In a cohort of 526 breast cancer patients, HORMAD1 was overexpressed in 71% of TNBC. In a second cohort of 186 TNBC patients treated with AC, HORMAD1 expression was associated with longer metastasis-free survival (MFS). In summary, HORMAD1 overexpression was predictive of an improved response to AC in PDX and is an independent prognostic factor in TNBC patients treated with AC.

PLK1 inhibition exhibits strong anti-tumoral activity in CCND1-driven breast cancer metastases with acquired palbociclib resistance.

A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

Localization and characterization of thyroid microcalcifications: A histopathological study.

Thyroid calcification is frequent in thyroid nodules. The aim of our study was to evaluate the prevalence of calcifications in thyroid tissue samples of patients with various thyroid diseases, and to identify their composition according to their localization. Among 50 thyroid samples included, 56% were malignant (papillary carcinoma) and 44% were benign (adenoma, multinodular goiter, Graves' disease, sarcoidosis). Calcifications were found in 95% of samples using polarised light microscopy, whereas only 12% were described in initial pathological reports. Three types were individualised and analyzed by infrared spectrometry (µFTIR): colloid calcifications composed of calcium oxalate, capsular calcifications and psammoma bodies, both composed of calcium phosphate. Of notice, psammoma bodies characterized by FE-SEM were composed of concentric structure suggesting a slow process for crystal deposition. Calcium phosphates were found only in malignant samples whereas calcium oxalate was not associated with a define pathology. Proliferation assessed by KI67 staining was high (33% of positive follicles), and RUNX2, OPN, and CD44 positive staining were detected in thyrocytes with a broad variation between samples. However, thyrocyte proliferation and differentiation markers were not associated with the number of crystals. TRPV5 and CaSR expression was also detected in thyrocytes. mRNA transcripts expression was confirmed in a subgroup of 10 patients, altogether with other calcium transporters such as PMCA1 or Cav1.3. Interestingly, TRPV5 mRNA expression was significantly associated with number of colloid calcifications (rho = -0.72; p = 0.02). The high prevalence of calcium oxalate crystals within colloid gel raises intriguing issues upon follicle physiology for calcium and oxalate transport.

Protein interacting with Amyloid Precursor Protein tail-1 (PAT1) is involved in early endocytosis.

Protein interacting with Amyloid Precursor Protein (APP) tail 1 (PAT1) also called APPBP2 or Ara 67 has different targets such as APP or androgen receptor and is expressed in several tissues. PAT1 is known to be involved in the subcellular trafficking of its targets. We previously observed in primary neurons that PAT1 is poorly associated with APP at the cell surface. Here we show that PAT1 colocalizes with vesicles close to the cell surface labeled with Rab5, Rab4, EEA1 and Rabaptin-5 but not with Rab11 and Rab7. Moreover, PAT1 expression regulates the number of EEA1 and Rab5 vesicles, and endocytosis/recycling of the transferrin receptor. In addition, low levels of PAT1 decrease the size of transferrin-colocalized EEA1 vesicles with time following transferrin uptake. Finally, overexpression of the APP binding domain to PAT1 is sufficient to compromise endocytosis. Altogether, these data suggest that PAT1 is a new actor in transferrin early endocytosis. Whether this new function of PAT1 may have consequences in pathology remains to be determined.

Stiripentol protects against calcium oxalate nephrolithiasis and ethylene glycol poisoning.

Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzymes deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome, possibly by inhibiting neuronal lactate dehydrogenase 5 isoenzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that Stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, Stiripentol decreased in a dose-dependent manner the synthesis of oxalate by hepatocytes. In vivo, Stiripentol oral administration reduced significantly urine oxalate excretion in rats. Stiripentol protected kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, Stiripentol improved significantly renal function. Patients affected by Dravet syndrome and treated with Stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received Stiripentol for several weeks: urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.

A large collection of integrated genomically characterized patient-derived xenografts highlighting the heterogeneity of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.

Urothelium proliferation is a trigger for renal crystal deposits in a murine lithogenesis model.

Most mouse kidney stone models induce nephrocalcinosis rather than urolithiasis. The aim of our study was to find an accelerated experimental model in order to study the early events of stone formation, that is, at the time of crystal binding to intrarenal urothelium. C57B6 mice exposed to vitamin D supplements and water containing hydroxyl-L-proline, ammonium chloride and calcium chloride were studied for 42 days. A group receiving urothelial cell mitogen Fibroblast Growth Factor 7 (FGF7) was compared to control group receiving saline. Calcium oxalate monohydrate (COM) crystals were detected in urines by day 2 and within urinary spaces in specialized fornix areas in both groups as soon as day 14 with enhanced deposits in FGF7 group compared to controls at day 21. Urothelial cells proliferation, uroplakin III downregulation and de novo expression of osteopontin receptor CD44 detected in FGF7 group, were delayed in the control group (day 42). Crystal aggregates within specialized fornix areas by day 42 were located in urinary spaces but also within and under a multilayered metaplastic urothelium, simultaneous to macrophages influx. Point of note, administration of a normal diet by day 21 was responsible for a spontaneous crystal clearance. Our data show that under supersaturation conditions, urothelial cell proliferation and calcium oxalate crystal retention occur within specialized fornix areas. Enhanced crystal deposits following FGF7 administration suggest that urothelium proliferation would be a relevant trigger for renal stone formation.

ABCC6 Deficiency Promotes Development of Randall Plaque.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a genetic disease caused by mutations in the ABCC6 gene that result in low pyrophosphate levels and subsequent progressive soft tissue calcifications. PXE mainly affects the skin, retina, and arteries. However, many patients with PXE experience kidney stones. We determined the prevalence of this pathology in patients with PXE and examined the possible underlying mechanisms in murine models. METHODS: We conducted a retrospective study in a large cohort of patients with PXE and analyzed urine samples and kidneys from Abcc6-/- mice at various ages. We used Yasue staining, scanning electron microscopy, electron microscopy coupled to electron energy loss spectroscopy, and Fourier transform infrared microspectroscopy to characterize kidney calcifications. RESULTS: Among 113 patients with PXE, 45 (40%) had a past medical history of kidney stones. Five of six computed tomography scans performed showed evidence of massive papillary calcifications (Randall plaques). Abcc6-/- mice spontaneously developed kidney interstitial apatite calcifications with aging. These calcifications appeared specifically at the tip of the papilla and formed Randall plaques similar to those observed in human kidneys. Compared with controls, Abcc6-/- mice had low urinary excretion of pyrophosphate. CONCLUSIONS: The frequency of kidney stones and probably, Randall plaque is extremely high in patients with PXE, and Abcc6-/- mice provide a new and useful model in which to study Randall plaque formation. Our findings also suggest that pyrophosphate administration should be evaluated for the prevention of Randall plaque and kidney stones.

High frequency and wide range of human kidney papillary crystalline plugs.

Most of kidney stones are supposed to originate from Randall's plaque at the tip of the papilla or from papillary tubular plugs. Nevertheless, the frequency and the composition of crystalline plugs remain only partly described. The objective was to assess the frequency, the composition and the topography of papillary plugs in human kidneys. A total of 76 papillae from 25 kidneys removed for cancer and without stones were analysed by immunohistochemistry combined with Yasue staining, field emission-scanning electron microscopy and Fourier transformed infrared micro-spectroscopy. Papillary tubular plugs have been observed by Yasue staining in 23/25 patients (92%) and 52/76 papillae (68%). Most of these plugs were made of calcium phosphate, mainly carbonated apatite and amorphous calcium phosphate, and rarely octacalcium phosphate pentahydrate. Calcium and magnesium phosphate (whitlockite) have also been observed. Based upon immunostaining coupled to Yasue coloration, most of calcium phosphate plugs were located in the deepest part of the loop of Henle. Calcium oxalate monohydrate and dihydrate tubular plugs were less frequent and stood in collecting ducts. At last, we observed calcium phosphate plugs deforming and sometimes breaking adjacent collecting ducts. Papillary tubular plugging, which may be considered as a potential first step toward kidney stone formation, is a very frequent setting, even in kidneys of non-stone formers. The variety in their composition and the distal precipitation of calcium oxalate suggest that plugs may occur in various conditions of urine supersaturation. Plugs were sometimes associated with collecting duct deformation.

Calcium and vitamin D have a synergistic role in a rat model of kidney stone disease.

Vitamin D supplementation in humans should be accompanied by calcium administration to avoid bone demineralization through vitamin D receptor signaling. Here we analyzed whether long-term exposure of rats to vitamin D supplementation, with or without a calcium-rich diet, would promote kidney stone formation. Four groups of rats received vitamin D alone (100,000 UI/kg/3 weeks), a calcium-enriched diet alone, both vitamin D supplementation and calcium-enriched diet, or a standard diet (controls) for 6 months. Serum and urine parameters and crystalluria were monitored. Kidney stones were assessed by 3-dimensional micro-computed tomography, infrared spectroscopy, von Kossa/Yasue staining, and field emission scanning electron microscopy. Although serum calcium levels were similar in the 4 groups, rats receiving vitamin D had a progressive increase in urinary calcium excretion over time, especially those receiving both calcium and vitamin D. However, oral calcium supplementation alone did not increase urinary calcium excretion. At 6 months, rats exposed to both calcium and vitamin D, but not rats exposed to calcium or vitamin D alone, developed significant apatite kidney calcifications (mean volume, 0.121 mm(3)). Thus, coadministration of vitamin D and increased calcium intake had a synergistic role in tubular calcifications or kidney stone formation in this rat model. Hence, one should be cautious about the cumulative risk of kidney stone formation in humans when exposed to both vitamin D supplementation and high calcium intake.

Topography, Composition and Structure of Incipient Randall Plaque at the Nanoscale Level.

PURPOSE: Randall identified calcium phosphate plaques in renal papillae as the origin of kidney stones. However, little is known about the early steps of Randall plaque formation preceding the onset of urolithiasis. Our objective was to characterize the composition and the initial formation site of incipient Randall plaque in nonstone forming, living patients. MATERIALS AND METHODS: Median patient age was 67.7 years. A total of 54 healthy papillae from kidneys removed for cancer and without stones were analyzed by immunohistochemistry and von Kossa staining, field emission-scanning electron microscopy with energy dispersive x-ray analysis, µ-Fourier transform infrared spectroscopy, cryo-transmission electron microscopy coupled to selected area electron diffraction and electron energy loss spectroscopy. RESULTS: Incipient Randall plaque was observed in 72.7% of kidneys. As expected, carbonated apatite was the main component of microcalcifications but amorphous calcium phosphate and whitlockite were identified in 80% and 40% of papillae, respectively. Incipient plaques were noted in the deepest part of the papillae around the loop of Henle tip as well as around the vasa recta, representing 62.4% and 37.2% of microcalcifications, respectively. Plaques were rarely close to collecting ducts. At the nanoscale level incipient calcifications were often composed of several nanocrystals in organic material that looked like microvesicles. CONCLUSIONS: Incipient Randall plaque is frequent. It appears not only at the extreme tip of the renal papillae around the hairpin structure of the loop of Henle but also around the vasa recta. Nanoscale analyses suggest a local nucleation process promoting nanocrystal growth in a supersaturated milieu. In addition, plaques contain various calcium and magnesium phosphates, and not only carbonated apatite.

PAT1 inversely regulates the surface Amyloid Precursor Protein level in mouse primary neurons.

BACKGROUND: The amyloid precursor protein (APP) is a key molecule in Alzheimer disease. Its localization at the cell surface can trigger downstream signaling and APP cleavages. APP trafficking to the cell surface in neurons is not clearly understood and may be related to the interactions with its partners. In this respect, by having homologies with kinesin light chain domains and because of its capacity to bind APP, PAT1 represents a good candidate. RESULTS: We observed that PAT1 binds poorly APP at the cell surface of primary cortical neurons contrary to cytoplasmic APP. Using down and up-regulation of PAT1, we observed respectively an increase and decrease of APP at the cell surface. The increase of APP at the cell surface induced by low levels of PAT1 did not trigger cell death signaling. CONCLUSIONS: These data suggest that PAT1 slows down APP trafficking to the cell surface in primary cortical neurons. Our results contribute to the elucidation of mechanisms involved in APP trafficking in Alzheimer disease.

Cytoplasmic SET induces tau hyperphosphorylation through a decrease of methylated phosphatase 2A.

BACKGROUND: The neuronal cytoplasmic localization of SET, an inhibitor of the phosphatase 2A (PP2A), results in tau hyperphosphorylation in the brains of Alzheimer patients through mechanisms that are still not well defined. RESULTS: We used primary neurons and mouse brain slices to show that SET is translocated to the cytoplasm in a manner independent of both its cleavage and over-expression. The localization of SET in the cytoplasm, either by the translocation of endogenous SET or by internalization of the recombinant full-length SET protein, induced tau hyperphosphorylation. Cytoplasmic recombinant full-length SET in mouse brain slices induced a decrease of PP2A activity through a decrease of methylated PP2A levels. The levels of methylated PP2A were negatively correlated with tau hyperphosphorylation at Ser-202 but not with the abnormal phosphorylation of tau at Ser-422. CONCLUSIONS: The presence of full-length SET in the neuronal cytoplasm is sufficient to impair PP2A methylation and activity, leading to tau hyperphosphorylation. In addition, our data suggest that tau hyperphosphorylation is regulated by different mechanisms at distinct sites. The translocation of SET to the neuronal cytoplasm, the low activity of PP2A, and tau hyperphosphorylation are associated in the brains of Alzheimer patients. Our data show a link between the translocation of SET in the cytoplasm and the decrease of methylated PP2A levels leading to a decrease of PP2A activity and tau hyperphosphorylation. This chain of events may contribute to the pathogenesis of Alzheimer disease.