Multi-omics profiling reveals dysregulated ribosome biogenesis and impaired cell proliferation following knockout of CDR2L.

BACKGROUND: Cerebellar degeneration-related (CDR) proteins are associated with paraneoplastic cerebellar degeneration (PCD) - a rare, neurodegenerative disease caused by tumour-induced autoimmunity against neural antigens resulting in degeneration of Purkinje neurons in the cerebellum. The pathogenesis of PCD is unknown, in large part due to our limited understanding of the functions of CDR proteins. To this end, we performed an extensive, multi-omics analysis of CDR-knockout cells focusing on the CDR2L protein, to gain a deeper understanding of the properties of the CDR proteins in ovarian cancer. METHODS: Ovarian cancer cell lines lacking either CDR1, CDR2, or CDR2L were analysed using RNA sequencing and mass spectrometry-based proteomics to assess changes to the transcriptome, proteome and secretome in the absence of these proteins. RESULTS: For each knockout cell line, we identified sets of differentially expressed genes and proteins. CDR2L-knockout cells displayed a distinct expression profile compared to CDR1- and CDR2-knockout cells. Knockout of CDR2L caused dysregulation of genes involved in ribosome biogenesis, protein translation, and cell cycle progression, ultimately causing impaired cell proliferation in vitro. Several of these genes showed a concurrent upregulation at the transcript level and downregulation at the protein level. CONCLUSIONS: Our study provides the first integrative multi-omics analysis of the impact of knockout of the CDR genes, providing both new insights into the biological properties of the CDR proteins in ovarian cancer, and a valuable resource for future investigations into the CDR proteins.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

The effects of phenylalanine and tyrosine levels on dopamine production in rat PC12 cells. Implications for treatment of phenylketonuria, tyrosinemia type 1 and comorbid neurodevelopmental disorders.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in phenylalanine accumulation and impaired tyrosine production. In Tyrosinemia type 1 (TYRSN1) mutations affect fumarylacetoacetate hydrolase, leading to accumulation of toxic intermediates of tyrosine catabolism. Treatment of TYRSN1 with nitisinone results in extreme tissue levels of tyrosine. Although PKU and TYRSN1 have opposite effects on tyrosine levels, both conditions have been associated with neuro-psychiatric symptoms typically present in ADHD, possibly indicating an impaired dopamine (DA) synthesis. However, concrete in vivo data on the possible molecular basis for disrupted DA production under disease mimicking conditions have been lacking. In pursuit to uncover associated molecular mechanisms, we exposed an established, DA producing cell line (PC12) to different concentrations of phenylalanine and tyrosine in culture media. We measured the effects on viability, proteomic composition, tyrosine, DA and tyrosine hydroxylase (TH) levels and TH phosphorylation. TH catalyzes the rate-limiting step in DA synthesis. High extracellular levels of phenylalanine depleted cells of intracellular tyrosine and DA. Compared to physiological levels (75 µM), either low (35 µM) or high concentrations of tyrosine (275 or 835 µM) decreased cellular DA, TH protein, and its phosphorylation levels. Using deep proteomic analysis, we identified multiple proteins, biological processes and pathways that were altered, including enzymes and transporters involved in amino acid metabolism. Using this information and published data, we developed a mathematical model to predict how extracellular levels of aromatic amino acids can affect the cellular synthesis of DA via different mechanisms. Together, these data provide new information about the normal regulation of neurotransmitter synthesis and how this may be altered in neurometabolic disorders, such as PKU and TYRSN1, with implications for the treatment of cognitive symptoms resulting from comorbid neurodevelopmental disorders.

Proteomic analysis unveils Gb3-independent alterations and mitochondrial dysfunction in a gla-/- zebrafish model of Fabry disease.

BACKGROUND: Fabry disease (FD) is a rare lysosomal storage disorder caused by mutations in the GLA gene, resulting in reduced or lack of α-galactosidase A activity. This results in the accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in lysosomes causing cellular impairment and organ failures. While current therapies focus on reversing Gb3 accumulation, they do not address the altered cellular signaling in FD. Therefore, this study aims to explore Gb3-independent mechanisms of kidney damage in Fabry disease and identify potential biomarkers. METHODS: To investigate these mechanisms, we utilized a zebrafish (ZF) gla-/- mutant (MU) model. ZF naturally lack A4GALT gene and, therefore, cannot synthesize Gb3. We obtained kidney samples from both wild-type (WT) (n = 8) and MU (n = 8) ZF and conducted proteome profiling using untargeted mass spectrometry. Additionally, we examined mitochondria morphology and cristae morphology using electron microscopy. To assess oxidative stress, we measured total antioxidant activity. Finally, immunohistochemistry was conducted on kidney samples to validate specific proteins. RESULTS: Our proteomics analysis of renal tissues from zebrafish revealed downregulation of lysosome and mitochondrial-related proteins in gla-/- MU renal tissues, while energy-related pathways including carbon, glycolysis, and galactose metabolisms were disturbed. Moreover, we observed abnormal mitochondrial shape, disrupted cristae morphology, altered mitochondrial volume and lower antioxidant activity in gla-/- MU ZF. CONCLUSIONS: These results suggest that the alterations observed at the proteome and mitochondrial level closely resemble well-known GLA mutation-related alterations in humans. Importantly, they also unveil novel Gb3-independent pathogenic mechanisms in Fabry disease. Understanding these mechanisms could potentially lead to the development of innovative drug screening approaches. Furthermore, the findings pave the way for identifying new clinical targets, offering new avenues for therapeutic interventions in Fabry disease. The zebrafish gla-/- mutant model proves valuable in elucidating these mechanisms and may contribute significantly to advancing our knowledge of this disorder.

Hypoxia induced responses are reflected in the stromal proteome of breast cancer.

Cancers are often associated with hypoxia and metabolic reprogramming, resulting in enhanced tumor progression. Here, we aim to study breast cancer hypoxia responses, focusing on secreted proteins from low-grade (luminal-like) and high-grade (basal-like) cell lines before and after hypoxia. We examine the overlap between proteomics data from secretome analysis and laser microdissected human breast cancer stroma, and we identify a 33-protein stromal-based hypoxia profile (33P) capturing differences between luminal-like and basal-like tumors. The 33P signature is associated with metabolic differences and other adaptations following hypoxia. We observe that mRNA values for 33P predict patient survival independently of molecular subtypes and basic prognostic factors, also among low-grade luminal-like tumors. We find a significant prognostic interaction between 33P and radiation therapy.

Proteomic analysis of peripheral blood mononuclear cells isolated from patients with pulmonary tuberculosis: A pilot study from Zanzibar, Tanzania.

This study aimed at exploring the proteomic profile of PBMCs to predict treatment response in pulmonary tuberculosis (PTB). This was a pilot study conducted among 8 adult patients from Zanzibar, Tanzania with confirmed PTB. Blood samples were collected at baseline, at 2 months of treatment, and at the end of treatment at 6 months. Proteins were extracted from PBMCs and analyzed using LC-MS/MS based label free quantitative proteomics. Overall, 3,530 proteins were quantified across the samples, and 12 differentially expressed proteins were identified at both 2 months of treatment and at treatment completion, which were involved in cellular and metabolic processes, as well as binding and catalytic activity. Seven were downregulated proteins (HSPA1B/HSPA1A, HSPH1, HSP90AA1, lipopolysaccharide-binding protein, complement component 9, calcyclin-binding protein, and protein transport protein Sec31A), and 5 proteins were upregulated (SEC14 domain and spectrin repeat-containing protein 1, leucine-rich repeat-containing 8 VRAC subunit D, homogentisate 1,2-dioxygenase, NEDD8-activating enzyme E1 regulatory subunit, and N-acetylserotonin O-methyltransferase-like protein). The results showed that proteome analysis of PBMCs can be used as a novel technique to identify protein abundance change with anti-tuberculosis treatment. The novel proteins elucidated in this work may provide new insights for understanding PTB pathogenesis, treatment, and prognosis.

Early response evaluation by single cell signaling profiling in acute myeloid leukemia.

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phospho-ERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.

Quantitative proteome profiling reveals molecular hallmarks of egg quality in Atlantic halibut: impairments of transcription and protein folding impede protein and energy homeostasis during early development.

BACKGROUND: Tandem mass tag spectrometry (TMT labeling-LC-MS/MS) was utilized to examine the global proteomes of Atlantic halibut eggs at the 1-cell-stage post fertilization. Comparisons were made between eggs judged to be of good quality (GQ) versus poor quality (BQ) as evidenced by their subsequent rates of survival for 12 days. Altered abundance of selected proteins in BQ eggs was confirmed by parallel reaction monitoring spectrometry (PRM-LC-MS/MS). Correspondence of protein levels to expression of related gene transcripts was examined via qPCR. Potential mitochondrial differences between GQ and BQ eggs were assessed by transmission electron microscopy (TEM) and measurements of mitochondrial DNA (mtDNA) levels. RESULTS: A total of 115 proteins were found to be differentially abundant between GQ and BQ eggs. Frequency distributions of these proteins indicated higher protein folding activity in GQ eggs compared to higher transcription and protein degradation activities in BQ eggs. BQ eggs were also significantly enriched with proteins related to mitochondrial structure and biogenesis. Quantitative differences in abundance of several proteins with parallel differences in their transcript levels were confirmed in egg samples obtained over three consecutive reproductive seasons. The observed disparities in global proteome profiles suggest impairment of protein and energy homeostasis related to unfolded protein response and mitochondrial stress in BQ eggs. TEM revealed BQ eggs to contain significantly higher numbers of mitochondria, but differences in corresponding genomic mtDNA (mt-nd5 and mt-atp6) levels were not significant. Mitochondria from BQ eggs were significantly smaller with a more irregular shape and a higher number of cristae than those from GQ eggs. CONCLUSION: The results of this study indicate that BQ Atlantic halibut eggs are impaired at both transcription and translation levels leading to endoplasmic reticulum and mitochondrial disorders. Observation of these irregularities over three consecutive reproductive seasons in BQ eggs from females of diverse background, age and reproductive experience indicates that they are a hallmark of poor egg quality. Additional research is needed to discover when in oogenesis and under what circumstances these defects may arise. The prevalence of this suite of markers in BQ eggs of diverse vertebrate species also begs investigation.

TGF-ß promotes microtube formation in glioblastoma through thrombospondin 1.

BACKGROUND: Microtubes (MTs), cytoplasmic extensions of glioma cells, are important cell communication structures promoting invasion and treatment resistance through network formation. MTs are abundant in chemoresistant gliomas, in particular, glioblastomas (GBMs), while they are uncommon in chemosensitive IDH-mutant and 1p/19q co-deleted oligodendrogliomas. The aim of this study was to identify potential signaling pathways involved in MT formation. METHODS: Bioinformatics analysis of TCGA was performed to analyze differences between GBM and oligodendroglioma. Patient-derived GBM stem cell lines were used to investigate MT formation under transforming growth factor-beta (TGF-ß) stimulation and inhibition in vitro and in vivo in an orthotopic xenograft model. RNA sequencing and proteomics were performed to detect commonalities and differences between GBM cell lines stimulated with TGF-ß. RESULTS: Analysis of TCGA data showed that the TGF-ß pathway is highly activated in GBMs compared to oligodendroglial tumors. We demonstrated that TGF-ß1 stimulation of GBM cell lines promotes enhanced MT formation and communication via calcium signaling. Inhibition of the TGF-ß pathway significantly reduced MT formation and its associated invasion in vitro and in vivo. Downstream of TGF-ß, we identified thrombospondin 1 (TSP1) as a potential mediator of MT formation in GBM through SMAD activation. TSP1 was upregulated upon TGF-ß stimulation and enhanced MT formation, which was inhibited by TSP1 shRNAs in vitro and in vivo. CONCLUSION: TGF-ß and its downstream mediator TSP1 are important mediators of the MT network in GBM and blocking this pathway could potentially help to break the complex MT-driven invasion/resistance network.

Bortezomib abrogates temozolomide-induced autophagic flux through an ATG5 dependent pathway.

Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis. Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro. We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5-/- (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery. Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival. Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway.

Patient Heterogeneity in Acute Myeloid Leukemia: Leukemic Cell Communication by Release of Soluble Mediators and Its Effects on Mesenchymal Stem Cells.

Acute myeloid leukemia (AML) is an aggressive bone marrow malignancy, and non-leukemic stromal cells (including mesenchymal stem cells, MSCs) are involved in leukemogenesis and show AML-supporting effects. We investigated how constitutive extracellular mediator release by primary human AML cells alters proteomic profiles of normal bone marrow MSCs. An average of 6814 proteins (range 6493-6918 proteins) were quantified for 41 MSC cultures supplemented with AML-cell conditioned medium, whereas an average of 6715 proteins (range 6703-6722) were quantified for untreated control MSCs. The AML effect on global MSC proteomic profiles varied between patients. Hierarchical clustering analysis identified 10 patients (5/10 secondary AML) showing more extensive AML-effects on the MSC proteome, whereas the other 31 patients clustered together with the untreated control MSCs and showed less extensive AML-induced effects. These two patient subsets differed especially with regard to MSC levels of extracellular matrix and mitochondrial/metabolic regulatory proteins. Less than 10% of MSC proteins were significantly altered by the exposure to AML-conditioned media; 301 proteins could only be quantified after exposure to conditioned medium and 201 additional proteins were significantly altered compared with the levels in control samples (153 increased, 48 decreased). The AML-modulated MSC proteins formed several interacting networks mainly reflecting intracellular organellar structure/trafficking but also extracellular matrix/cytokine signaling, and a single small network reflecting altered DNA replication. Our results suggest that targeting of intracellular trafficking and/or intercellular communication is a possible therapeutic strategy in AML.

Proteomic Comparison of Bone Marrow Derived Osteoblasts and Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, and therapeutic targeting of these cells is considered both for malignant and non-malignant diseases. We analyzed global proteomic profiles for osteoblasts derived from ten and MSCs from six healthy individuals, and we quantified 5465 proteins for the osteoblasts and 5420 proteins for the MSCs. There was a large overlap in the profiles for the two cell types; 156 proteins were quantified only in osteoblasts and 111 proteins only for the MSCs. The osteoblast-specific proteins included several extracellular matrix proteins and a network including 27 proteins that influence intracellular signaling (Wnt/Notch/Bone morphogenic protein pathways) and bone mineralization. The osteoblasts and MSCs showed only minor age- and sex-dependent proteomic differences. Finally, the osteoblast and MSC proteomic profiles were altered by ex vivo culture in serum-free media. We conclude that although the proteomic profiles of osteoblasts and MSCs show many similarities, we identified several osteoblast-specific extracellular matrix proteins and an osteoblast-specific intracellular signaling network. Therapeutic targeting of these proteins will possibly have minor effects on MSCs. Furthermore, the use of ex vivo cultured osteoblasts/MSCs in clinical medicine will require careful standardization of the ex vivo handling of the cells.

The Constitutive Extracellular Protein Release by Acute Myeloid Leukemia Cells-A Proteomic Study of Patient Heterogeneity and Its Modulation by Mesenchymal Stromal Cells.

Extracellular protein release is important both for the formation of extracellular matrix and for communication between cells. We investigated the extracellular protein release by in vitro cultured normal mesenchymal stem cells (MSCs) and by primary human acute myeloid leukemia (AML) cells derived from 40 consecutive patients. We observed quantifiable levels of 3082 proteins in our study; for the MSCs, we detected 1446 proteins, whereas the number of released proteins for the AML cells showed wide variation between patients (average number 1699, range 557-2380). The proteins were derived from various cellular compartments (e.g., cell membrane, nucleus, and cytoplasms), several organelles (e.g., cytoskeleton, endoplasmatic reticulum, Golgi apparatus, and mitochondria) and had various functions (e.g., extracellular matrix and exosomal proteins, cytokines, soluble adhesion molecules, protein synthesis, post-transcriptional modulation, RNA binding, and ribonuclear proteins). Thus, AML patients were very heterogeneous both regarding the number of proteins and the nature of their extracellularly released proteins. The protein release profiles of MSCs and primary AML cells show a considerable overlap, but a minority of the proteins are released only or mainly by the MSC, including several extracellular matrix molecules. Taken together, our observations suggest that the protein profile of the extracellular bone marrow microenvironment differs between AML patients, these differences are mainly caused by the protein release by the leukemic cells but this leukemia-associated heterogeneity of the overall extracellular protein profile is modulated by the constitutive protein release by normal MSCs.

The Extracellular Bone Marrow Microenvironment-A Proteomic Comparison of Constitutive Protein Release by In Vitro Cultured Osteoblasts and Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) and osteoblasts are bone marrow stromal cells that contribute to the formation of stem cell niches and support normal hematopoiesis, leukemogenesis and development of metastases from distant cancers. This support is mediated through cell-cell contact, release of soluble mediators and formation of extracellular matrix. By using a proteomic approach, we characterized the protein release by in vitro cultured human MSCs (10 donors) and osteoblasts (nine donors). We identified 1379 molecules released by these cells, including 340 proteins belonging to the GO-term Extracellular matrix. Both cell types released a wide range of functionally heterogeneous proteins including extracellular matrix molecules (especially collagens), several enzymes and especially proteases, cytokines and soluble adhesion molecules, but also several intracellular molecules including chaperones, cytoplasmic mediators, histones and non-histone nuclear molecules. The levels of most proteins did not differ between MSCs and osteoblasts, but 82 proteins were more abundant for MSC (especially extracellular matrix proteins and proteases) and 36 proteins more abundant for osteoblasts. Finally, a large number of exosomal proteins were identified. To conclude, MSCs and osteoblasts show extracellular release of a wide range of functionally diverse proteins, including several extracellular matrix molecules known to support cancer progression (e.g., metastases from distant tumors, increased relapse risk for hematological malignancies), and the large number of identified exosomal proteins suggests that exocytosis is an important mechanism of protein release.

Stathmin expression associates with vascular and immune responses in aggressive breast cancer subgroups.

Studies indicate that stathmin expression associates with PI3K activation in breast cancer, suggesting stathmin as a marker for targetable patient subgroups. Here we assessed stathmin in relation to tumour proliferation, vascular and immune responses, BRCA1 germline status, basal-like differentiation, clinico-pathologic features, and survival. Immunohistochemical staining was performed on breast cancers from two series (cohort 1, n = 187; cohort 2, n = 198), and mass spectrometry data from 24 cases and 12 breast cancer cell lines was examined for proteomic profiles. Open databases were also explored (TCGA, METABRIC, Oslo2 Landscape cohort, Cancer Cell Line Encyclopedia). High stathmin expression associated with tumour proliferation, p53 status, basal-like differentiation, BRCA1 genotype, and high-grade histology. These patterns were confirmed using mRNA data. Stathmin mRNA further associated with tumour angiogenesis, immune responses and reduced survival. By logistic regression, stathmin protein independently predicted a BRCA1 genotype (OR 10.0, p = 0.015) among ER negative tumours. Cell line analysis (Connectivity Map) implied PI3K inhibition in tumours with high stathmin. Altogether, our findings indicate that stathmin might be involved in the regulation of tumour angiogenesis and immune responses in breast cancer, in addition to tumour proliferation. Cell data point to potential effects of PI3K inhibition in tumours with high stathmin expression.

PIK3CA Amplification Associates with Aggressive Phenotype but Not Markers of AKT-MTOR Signaling in Endometrial Carcinoma.

PURPOSE: Amplification of PIK3CA, encoding the PI3K catalytic subunit alpha, is common in uterine corpus endometrial carcinoma (UCEC) and linked to an aggressive phenotype. However, it is unclear whether PIK3CA amplification acts via PI3K activation. We investigated the association between PIK3CA amplification, markers of PI3K activity, and prognosis in a large cohort of UCEC specimens. EXPERIMENTAL DESIGN: UCECs from 591 clinically annotated patients including 83 tumors with matching metastasis (n = 188) were analyzed by FISH to determine PIK3CA copy-number status. These data were integrated with mRNA and protein expression and clinicopathologic data. Results were verified in The Cancer Genome Atlas dataset. RESULTS: PIK3CA amplifications were associated with disease-specific mortality and with other markers of aggressive disease. PIK3CA amplifications were also associated with other amplifications characteristic of the serous-like somatic copy-number alteration (SCNA)-high subgroup of UCEC. Tumors with PIK3CA amplification also demonstrated an increase in phospho-p70S6K but had decreased levels of activated phospho-AKT1-3 as assessed by Reverse Phase Protein Arrays and an mRNA signature of MTOR inhibition. CONCLUSIONS: PIK3CA amplification is a strong prognostic marker and a potential marker for the aggressive SCNA-high subgroup of UCEC. Although PIK3CA amplification associates with some surrogate measures of increased PI3K activity, markers for AKT1-3 and MTOR signaling are decreased, suggesting that this signaling is not a predominant pathway to promote cancer growth of aggressive serous-like UCEC. Moreover, these associations may reflect features of the SCNA-high subgroup of UCEC rather than effects of PIK3CA amplification itself.

PIK3CA exon9 mutations associate with reduced survival, and are highly concordant between matching primary tumors and metastases in endometrial cancer.

Mutations of the phosphoinositide-3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) are frequent in endometrial cancer. We sequenced exon9 and exon20 of PIK3CA in 280 primary endometrial cancers to assess the relationship with clinicopathologic variables, patient survival and associations with PIK3CA mRNA and phospho-AKT1 by gene expression and protein data, respectively. While PIK3CA mutations generally had no impact on survival, and were not associated with clinicopathological variables, patients with exon9 charge-changing mutations, providing a positive charge at the substituted amino acid residue, were associated with poor survival (p = 0.018). Furthermore, we characterized PIK3CA mutations in the metastatic setting, including 32 patients with matched primary tumors and metastases, and found a high level of concordance (85.7%; 6 out of 7 patients), suggesting limited heterogeneity. PIK3CA mRNA levels were increased in metastases compared to the primary tumors (p = 0.031), independent of PIK3CA mutation status, which rather associated with reduced PIK3CA mRNA expression. PIK3CA mutated tumors expressed higher p-AKT/AKT protein levels, both within primary (p < 0.001) and metastatic lesion (p = 0.010). Our results support the notion that the PI3K signaling pathway might be activated, both dependent- and independently of PIK3CA mutations, an aspect that should be considered when designing PIK3 pathway targeting strategies in endometrial cancer.