Combined PDGFR and HDAC Inhibition Overcomes PTEN Disruption in Chordoma

Background

The majority of chordomas show activation of the platelet-derived growth factor receptor (PDGFR). Based on in vitro intertumoral variation in response to recombinant PDGF protein and PDGFR inhibition, and variable tumor response to imatinib, we hypothesized that chordomas resistant to PDGFR inhibition may possess downstream activation of the pathway.

Methods

Molecular profiling was performed on 23 consecutive chordoma primary tissue specimens. Primary cultures established from 20 of the 23 specimens, and chordoma cell lines, UCH-1 and UCH-2, were used for in vitro experiments.

Results

Loss of heterozygosity (LOH) at the phosphatase and tensin homolog (PTEN) locus was observed in 6 specimens (26%). PTEN disruption statistically correlated with increased Ki-67 proliferation index, an established marker of poor outcome for chordoma. Compared to wild type, PTEN deficient chordomas displayed increased proliferative rate, and responded less favorably to PDGFR inhibition. PTEN gene restoration abrogated this growth advantage. Chordomas are characterized by intratumoral hypoxia and local invasion, and histone deacetylase (HDAC) inhibitors are capable of attenuating both hypoxic signaling and cell migration. The combination of PDGFR and HDAC inhibition effectively disrupted growth and invasion of PTEN deficient chordoma cells.

Conclusions

Loss of heterozygosity of the PTEN gene seen in a subset of chordomas is associated with aggressive in vitro behavior and strongly correlates with increased Ki-67 proliferative index. Combined inhibition of PDGFR and HDAC attenuates proliferation and invasion in chordoma cells deficient for PTEN.     

Genome-Wide Microarray Expression and Genomic Alterations by Array-CGH Analysis in Neuroblastoma Stem-Like Cells

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells.     

Overexpression of ERG and Wild-Type PTEN Are Associated with Favorable Clinical Prognosis and Low Biochemical Recurrence in Prostate Cancer

Objectives

The aim of this study was to investigate the expression of two commonly altered genes ERG and PTEN in prostate cancer (PC) and evaluate their prognostic significance. Despite conflicting published results, TMPRSS2-ERG gene fusion and PTEN loss are generally considered unfavorable markers for PC progression.

Materials and Methods

Of the 762 prostatic adenocarcinoma specimens obtained from radical prostatectomy, 613 without neoadjuvant hormone therapy were included in tissue microarrays for quantitatively assessment of ERG and PTEN expression via immunohistochemistry. Statistical analysis of the association between such expression and clinicopathological parameters, including clinical prognosis, was performed with a p-value of <0.05 considered significant.

Results

During a median follow-up period of 44.0 months, 132 (21.5%) patients developed biochemical recurrence (BCR). ERG overexpression and PTEN loss were observed in 145 (23.7%) and 253 (41.3%) cases, respectively. BCR-free survival was significantly better in patients with ERG overexpression (p=0.005), but unfavorable among those with PTEN loss (p=0.142). Sub-group analysis revealed that patients with PTEN loss and negative ERG expression had the worst BCR-free survival outcome (p=0.021). Furthermore, multivariate analysis identified prostate-specific antigen level (≥10 ng/mL), Gleason score (>6), pathologic T stage (≥T3), positive surgical margin, and extraprostatic capsule extension as significant risk factors for BCR (p<0.05).

Conclusions

Our results indicated that ERG overexpression was associated with favorable BCR-free survival after radical prostatectomy for PC, whereas PTEN loss was with unfavorable outcomes.     

Short Term Feeding of a High Fat Diet Exerts an Additive Effect on Hepatocellular Damage and Steatosis in Liver-Specific PTEN Knockout Mice

Background

Hepatospecific deletion of PTEN results in constitutive activation of Akt and increased lipogenesis. In mice, the addition of a high fat diet (HFD) downregulates lipogenesis. The aim of this study was to determine the effects of a HFD on hepatocellular damage induced by deletion of PTEN.

Methods

12 Week old male flox/flox hepatospecific PTEN mice (PTEN^f/f) or Alb-Cre controls were fed a HFD composed of 45% fat-derived calories (from corn oil) or a normal chow. Animals were then analyzed for hepatocellular damage, oxidative stress and expression of enzymes involved in fatty acid metabolism.

Results

In the Alb-Cre animals, the addition of a HFD resulted in a significant increase in liver triglycerides and altered REDOX capacity as evidenced by increased GPX activity, decreased GST activity and decreased hepatic concentrations of GSSG. In addition, SCD2, ACLY and FASN were all downregulated by the addition of HFD. Furthermore, expression of PPARα and PPARα-dependent proteins Cyp4a and ACSL1 were upregulated. In the PTEN^f/f mice, HFD resulted in significant increased in ALT, serum triglycerides and decreased REDOX capacity. Although expression of fatty acid synthetic enzymes was elevated in the chow fed PTEN^f/f group, the addition of HFD resulted in SCD2, ACLY and FASN downregulation. Compared to the Alb-Cre HFD group, expression of PGC1α, PPARα and its downstream targets ACSL and Cyp4a were upregulated in PTEN^f/f mice.

Conclusions

These data suggest that during conditions of constitutive Akt activation and increased steatosis, the addition of a HFD enhances hepatocellular damage due to increased CD36 expression and altered REDOX status. In addition, this work indicates HFD-induced hepatocellular damage occurs in part, independently of Akt signaling.     

Biomarkers of TGF-β Signaling Pathway and Prognosis of Pancreatic Cancer

Background

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

Method

We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

Results

The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

Conclusion

Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.     

Somatic Copy Number Abnormalities and Mutations in PI3K/AKT/mTOR Pathway Have Prognostic Significance for Overall Survival in Platinum Treated Locally Advanced or Metastatic Urothelial Tumors

Background

An integrative analysis was conducted to identify genomic alterations at a pathway level that could predict overall survival (OS) in patients with advanced urothelial carcinoma (UC) treated with platinum-based chemotherapy.

Patients and Methods

DNA and RNA were extracted from 103 formalin-fixed paraffin embedded (FFPE) invasive high-grade UC samples and were screened for mutations, copy number variation (CNV) and gene expression analysis. Clinical data were available from 85 cases. Mutations were analyzed by mass-spectrometry based on genotyping platform (Oncomap 3) and genomic imbalances were detected by comparative genomic hybridization (CGH) analysis. Regions with threshold of log² ratio ≥0.4, or ≤0.6 were defined as either having copy number gain or loss and significantly recurrent CNV across the set of samples were determined using a GISTIC analysis. Expression analysis on selected relevant UC genes was conducted using Nanostring. To define the co-occurrence pattern of mutations and CNV, we grouped genomic events into 5 core signal transduction pathways: 1) TP53 pathway, 2) RTK/RAS/RAF pathway, 3) PI3K/AKT/mTOR pathway, 4) WNT/CTNNB1, 5) RB1 pathway. Cox regression was used to assess pathways abnormalities with survival outcomes.

Results

35 samples (41%) harbored mutations on at least one gene: TP53 (16%), PIK3CA (9%), FGFR3 (2%), HRAS/KRAS (5%), and CTNNB1 (1%). 66% of patients had some sort of CNV. PIK3CA/AKT/mTOR pathway alteration (mutations+CNV) had the greatest impact on OS (p=0.055). At a gene level, overexpression of CTNNB1 (p=0.0008) and PIK3CA (p=0.02) were associated with shorter OS. Mutational status on PIK3CA was not associated with survival. Among other individually found genomic alterations, TP53 mutations (p=0.07), mTOR gain (p=0.07) and PTEN overexpression (p=0.08) have a marginally significant negative impact on OS.

Conclusions

Our study suggests that targeted therapies focusing on the PIK3CA/AKT/mTOR pathway genomic alterations can generate the greatest impact in the overall patient population of high-grade advanced UC.     

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Objective

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.

Methods

RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.

Results

The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).

Conclusions

RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.     

PTENε suppresses tumor metastasis through regulation of filopodia formation

PTEN is one of the most frequently mutated genes in malignancies and acts as a powerful tumor suppressor. Tumorigenesis is involved in multiple and complex processes including initiation, invasion, and metastasis. The complexity of PTEN function is partially attributed to PTEN family members such as PTENα and PTENβ. Here, we report the identification of PTENε (also named as PTEN5), a novel N‐terminal‐extended PTEN isoform that suppresses tumor invasion and metastasis. We show that the translation of PTENε/PTEN5 is initiated from the CUG⁸¹⁶ codon within the 5′UTR region of PTEN mRNA. PTENε/PTEN5 mainly localizes in the cell membrane and physically associates with and dephosphorylates VASP and ACTR2, which govern filopodia formation and cell motility. We found that endogenous depletion of PTENε/PTEN5 promotes filopodia formation and enhances the metastasis capacity of tumor cells. Overall, we identify a new isoform of PTEN with distinct subcellular localization and molecular function compared to the known members of the PTEN family. These findings advance our current understanding of the importance and diversity of PTEN functions.     

Multiple Functional Risk Variants in a SMAD7 Enhancer Implicate a Colorectal Cancer Risk Haplotype

Genome-wide association studies (GWAS) of colorectal cancer (CRC) have led to the identification of a number of common variants associated with modest risk. Several risk variants map within the vicinity of TGFβ/BMP signaling pathway genes, including rs4939827 within an intron of SMAD7 at 18q21.1. A previous study implicated a novel SNP (novel 1 or rs58920878) as a functional variant within an enhancer element in SMAD7 intron 4. In this study, we show that four SNPs including novel 1 (rs6507874, rs6507875, rs8085824, and rs58920878) in linkage disequilibrium (LD) with the index SNP rs4939827 demonstrate allele-specific enhancer effects in a large, multi-component enhancer of SMAD7. All four SNPs demonstrate allele-specific protein binding to nuclear extracts of CRC cell lines. Furthermore, some of the risk-associated alleles correlate with increased expression of SMAD7 in normal colon tissues. Finally, we show that the enhancer is responsive to BMP4 stimulation. Taken together, we propose that the associated CRC risk at 18q21.1 is due to four functional variants that regulate SMAD7 expression and potentially perturb a BMP negative feedback loop in TGFβ/BMP signaling pathways.     

High Prevalence and Clinical Relevance of Genes Affected by Chromosomal Breaks in Colorectal Cancer

Background

Cancer is caused by somatic DNA alterations such as gene point mutations, DNA copy number aberrations (CNA) and structural variants (SVs). Genome-wide analyses of SVs in large sample series with well-documented clinical information are still scarce. Consequently, the impact of SVs on carcinogenesis and patient outcome remains poorly understood. This study aimed to perform a systematic analysis of genes that are affected by CNA-associated chromosomal breaks in colorectal cancer (CRC) and to determine the clinical relevance of recurrent breakpoint genes.

Methods

Primary CRC samples of patients with metastatic disease from CAIRO and CAIRO2 clinical trials were previously characterized by array-comparative genomic hybridization. These data were now used to determine the prevalence of CNA-associated chromosomal breaks within genes across 352 CRC samples. In addition, mutation status of the commonly affected APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS genes was determined for 204 CRC samples by targeted massive parallel sequencing. Clinical relevance was assessed upon stratification of patients based on gene mutations and gene breakpoints that were observed in >3% of CRC cases.

Results

In total, 748 genes were identified that were recurrently affected by chromosomal breaks (FDR <0.1). MACROD2 was affected in 41% of CRC samples and another 169 genes showed breakpoints in >3% of cases, indicating that prevalence of gene breakpoints is comparable to the prevalence of well-known gene point mutations. Patient stratification based on gene breakpoints and point mutations revealed one CRC subtype with very poor prognosis.

Conclusions

We conclude that CNA-associated chromosomal breaks within genes represent a highly prevalent and clinically relevant subset of SVs in CRC.     

Investigation of <Emphasis Type="Italic">MMAC/PTEN</Emphasis> Gene Mutations and Protein Expression in Low Grade Gliomas

The MMAC/PTEN tumor suppressor gene has an essential biological role in the formation of glioblastomas. It is known that there are variations in genetic alterations in tumors that develop in patients with different ethnic backgrounds; thus, we aimed to evaluate the incidence of MMAC/PTEN mutations and protein expression among various low grade gliomas of Turkish patients. We investigated 28 low grade gliomas for mutations of the MMAC/PTEN gene using single strand conformational polymorphism method followed by DNA sequencing. Additionally, the level of MMAC/PTEN protein expression in the tissues of 26 tumors was assessed by immunohistochemistry. In our investigation, MMAC/PTEN mutations were detected in 2 of 28 tumors (7.14%). One novel sequence variant G → A transition at codon 159 was identified. This missense variation was a result of an alteration from AGG (Arginine) to AAG (Lysine). Moreover, it was observed that MMAC/PTEN protein expression was reduced to 73.08% of tumors. In conclusion, reduced MMAC/PTEN expression by genetic and/or epigenetic mechanisms in low grade gliomas might be associated with glioma tumorigenesis.     

Correlation between Activation of PI3K/AKT/mTOR Pathway and Prognosis of Breast Cancer in Chinese Women

Background

Abnormal activation of PI3K/AKT/mTOR (PAM) pathway, caused by PIK3CA mutation, KRAS mutation, PTEN loss, or AKT1 mutation, is one of the most frequent signaling abnormalities in breast carcinoma. However, distribution and frequencies of mutations in PAM pathway are unclear in breast cancer patients from the mainland of China and the correlation between these mutations and breast cancer outcome remains to be identified.

Methods

A total of 288 patients with invasive ductal breast cancer were recruited in this study. Mutations in PIK3CA (exons 4, 9 and 20), KRAS (exon 2) and AKT1 (exon 3) were detected using Sanger sequencing. PTEN loss was measured by immunohistochemistry assay. Correlations between these genetic aberrations and clinicopathological features were analyzed.

Results

The frequencies of PIK3CA mutation, KRAS mutation, AKT1 mutation and PTEN loss were 15.6%, 1.8%, 4.4% and 35.3%, respectively. However, except for PTEN loss, which was tied to estrogen receptor (ER) status, these alterations were not associated with other clinicopathological features. Survival analysis demonstrated that PIK3CA mutation, PTEN loss and PAM pathway activation were not associated with disease-free survival (DFS). Subgroup analysis of patients with ER positive tumors revealed that PIK3CA mutation more strongly reduced DFS compared to wild-type PIK3CA (76.2% vs. 54.2%; P = 0.011). PIK3CA mutation was also an independent factor for bad prognosis in ER positive patients.

Conclusions

AKT1, KRAS and PIK3CA mutations and PTEN loss all exist in women with breast cancer in the mainland China. PIK3CA mutation may contribute to the poor outcome of ER positive breast carcinomas, providing evidence for the combination of PI3K/AKT/mTOR inhibitors and endocrine therapy.     

Endothelial Lineage Differentiation from Induced Pluripotent Stem Cells Is Regulated by MicroRNA-21 and Transforming Growth Factor β2 (TGF-β2) Pathways

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In this paper, we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in predifferentiated iPSCs induced EC marker up-regulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong up-regulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21, and we showed that PTEN knockdown is required for miR-21-mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.     

Characterization of SMAD3 Gene Variants for Possible Roles in Ventricular Septal Defects and Other Congenital Heart Diseases

Background

Nodal/TGF signaling pathway has an important effect at early stages of differentiation of human embryonic stem cells in directing them to develop into different embryonic lineages. SMAD3 is a key intracellular messenger regulating factor in the Nodal/TGF signaling pathway, playing important roles in embryonic and, particularly, cardiovascular system development. The aim of this work was to find evidence on whether SMAD3 variations might be associated with ventricular septal defects (VSD) or other congenital heart diseases (CHD).

Methods

We sequenced the SMAD3 gene for 372 Chinese Han CHD patients including 176 VSD patients and evaluated SNP rs2289263, which is located before the 5’UTR sequence of the gene. The statistical analyses were conducted using Chi-Square Tests as implemented in SPSS (version 13.0). The Hardy-Weinberg equilibrium test of the population was carried out using the online software OEGE.

Results

Three heterozygous variants in SMAD3 gene, rs2289263, rs35874463 and rs17228212, were identified. Statistical analyses showed that the rs2289263 variant located before the 5’UTR sequence of SMAD3 gene was associated with the risk of VSD (P value=0.013 <0.05).

Conclusions

The SNP rs2289263 in the SMAD3 gene is associated with VSD in Chinese Han populations.     

Role of Vitamin D3 in Modulation of ΔNp63α Expression during UVB Induced Tumor Formation in SKH-1 Mice

ΔNp63α, a proto-oncogene, is up-regulated in non-melanoma skin cancers and directly regulates the expression of both Vitamin D receptor (VDR) and phosphatase and tensin homologue deleted on chromosome ten (PTEN). Since ΔNp63α has been shown to inhibit cell invasion via regulation of VDR, we wanted to determine whether dietary Vitamin D₃ protected against UVB induced tumor formation in SKH-1 mice, a model for squamous cell carcinoma development. We examined whether there was a correlation between dietary Vitamin D₃ and ΔNp63α, VDR or PTEN expression in vivo in SKH-1 mice chronically exposed to UVB radiation and fed chow containing increasing concentrations of dietary Vitamin D₃. Although we observed differential effects of the Vitamin D₃ diet on ΔNp63α and VDR expression in chronically irradiated normal mouse skin as well as UVB induced tumors, Vitamin D₃ had little effect on PTEN expression in vivo. While low-grade papillomas in mice exposed to UV and fed normal chow displayed increased levels of ΔNp63α, expression of both ΔNp63α and VDR was reduced in invasive tumors. Interestingly, in mice fed high Vitamin D₃ chow, elevated levels of ΔNp63α were observed in both local and invasive tumors but not in normal skin suggesting that oral supplementation with Vitamin D₃ may increase the proliferative potential of skin tumors by increasing ΔNp63α levels.