Bromodomain and extraterminal domain inhibitor enhances the antitumor effect of imatinib in gastrointestinal stromal tumours

In gastrointestinal stromal tumours (GISTs), the function of bromodomain‐containing 4 (BRD4) remains underexplored. BRD4 mRNA abundance was quantified in GISTs. In the current study, we investigated the role of BRD4 in GISTs. Our results show a significant enhancement in BRD4 mRNA and a shift from very low‐risk/low‐risk to high‐risk levels as per NCCN specifications. Overexpression of BRD4 correlated with unfavourable genotype, nongastric location, enhanced risk and decreased disease‐free survival, which were predicted independently. Knockout of BRD4 in vitro suppressed KIT expression, which led to inactivation of the KIT/PI3K/AKT/mTOR pathway, impeded migration and cell growth and made the resistant GIST cells sensitive to imatinib. The expression of KIT was repressed by a BRD4 inhibitor JQ1, which also induced myristoylated‐AKT‐suppressible caspases 3 and 9 activities, induced LC3‐II, exhibited dose‐dependent therapeutic synergy with imatinib and attenuated the activation of the PI3K/AKT/mTOR pathway. In comparison with their single therapy, the combination of JQ1/imatinib more efficiently suppressed the growth of xenografts and exhibited a reduction in KIT phosphorylation, a decrease in Ki‐67 and in the levels of phosphorylated PI3K/AKT/mTOR and enhanced TUNEL staining. Thus, we characterized the biological, prognostic and therapeutic implications of overexpressed BRD4 in GIST and observed that JQ1 suppresses KIT transactivation and nullifies the activation of PI3K/AKT/mTOR, providing a potential strategy for treating imatinib‐resistant GIST through dual blockade of KIT and BRD4.     

Kit K641E oncogene up-regulates Sprouty homolog 4 and Trophoblast glycoprotein in interstitial cells of Cajal in a murine model of gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the receptor tyrosine kinase KIT are present in most GIST. KIT K642E was originally identified in sporadic GIST and later found in the germ line of a familial GIST cohort. A mouse model harbouring a germline Kit K641E mutant was created to model familial GIST. The expression profile was investigated in the gastric antrum of the Kit ^K641E murine GIST model by microarray, quantitative PCR and immunofluorescence. Gja1/Cx43 , Gpc6 , Gpr133 , Pacrg , Pde3a , Prkar2b , Prkcq/Pkce , Rasd2 , Spry4 and Tpbg/5T4 were found to be up-regulated. The proteins encoded by Gja1/Cx43 , Pde3a , Prkcq/Pkce were localized in Kit-ir ICC in wild-type and Kit ^K641E animals while Spry4 and Tpbg/5T4 were detected in Kit-ir cells only in Kit ^K641E, but not in Kit ^WT/WT animals. Most up-regulated genes in this mouse model belong to the gene expression profile of human GIST but also to the profile of normal Kit⁺ ICC in the mouse small intestine. Spry4 and Tpbg/5T4 may represent candidates for targeted therapeutic approaches in GIST with oncogenic KIT mutations.     

miR-4510 acts as a tumor suppressor in gastrointestinal stromal tumor by targeting APOC2

Dysregulation of microRNAs (miRNAs) expression has been demonstrated in gastrointestinal stromal tumor (GIST). In this study, we aimed to determine the differential miRNAs expression in GISTs and explore the functional mechanism of differential miRNAs in GIST cells. We measured differential miRNAs in six pairs of GIST tissues and matched adjacent tissues through a high-throughput sequencing, which was confirmed in 64 pairs of GIST tissues and adjacent tissues by real-time polymerase chain reaction. We found that miR-4510 expression was significantly downregulated in GIST tissues compared to matched control tissues. Luciferase reporter assay identified apolipoprotein C-II (APOC2) as a direct target of miR-4510. Overexpression of miR-4510 inhibited the mRNA and protein expression of APOC2. In addition, overexpression of miR-4510 suppressed GIST cell proliferation, migration, and invasion. Overexpression of miR-4510 also inhibited the phosphorylation of AKT and ERK1/2, reduced the expression of matrix metallopeptidase 2 (MMP2) and MMP9. APOC2 knockdown mimicked the effect of miR-4510 overexpression. Further investigation confirmed that APOC2 was notably upregulated in GIST tissues compared to adjacent control tissues. These results suggested that miR-4510 downregulation could promote GIST progression, including growth, invasion, and metastasis, through increasing APOC2 expression.     

Prediction of aggressiveness of gastrointestinal stromal tumours based on immunostaining with bcl-2, Ki-67 and p53

OBJECTIVE: While the use of fine needle aspiration (FNA) in the diagnosis of gastrointestinal stromal tumours (GISTs) is well-established, it can be difficult to predict the prognosis of GIST based on morphology alone. The objective of the current study was to determine if expression of bcl-2, Ki-67 and p53 correlated with the outcome of GISTs based on cytological material. METHODS: Cell-blocks from 14 GISTs diagnosed by FNA were retrieved. Immunostaining was performed with antibodies against bcl-2, Ki-67 and p53. All cytological diagnoses were confirmed by positive immunostaining with c-kit and/or subsequent histological evaluation. Positivity for bcl-2, Ki-67 and p53 was defined as the presence of > or =10% cytoplasmic staining, > or =5% nuclear staining and > or =5% nuclear staining respectively. RESULTS: The 14 patients consisted of seven males and seven females with a mean age of 58 years. The average follow-up interval was 46 months. Six had a benign course and eight developed recurrences/metastases. Thirteen (93%) cases showed positive staining for bcl-2. Positive Ki-67 and p53 staining was noted in one (7%) and seven (50%) cases respectively. The difference in staining for p53 between aggressive and non-aggressive GISTs was statistically significant. No statistically significant difference was noted for bcl-2 staining or Ki-67 labelling index between the two groups. CONCLUSIONS: According to our observations, p53 immunostaining may be useful in predicting the outcome of GIST diagnosed by FNA; Ki-67 and bcl-2 are not useful as prognostic markers for GIST in FNA specimens.     

Clinicopathologic factors and nuclear morphometry as independent prognosticators in KIT-positive gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms found in the gastrointestinal tract. The purpose of this study was to evaluate whether morphometric measurements could complement tumor size and mitotic activity in risk evaluation. Nuclear roundness and ellipse axis ratio were found to correlate with tumor size, mitotic activity, nuclear atypia, and hemorrhage. Morphometric variables in 422 GISTs were significant for overall survival in univariate analyses but did not retain independent significance in multivariate analyses incorporating mitotic count and tumor size. Traditional variables, together with sex, location of primary tumor, and nuclear atypia, seem to be the best parameters for prognostic evaluation.     

MiR-320a downregulation is associated with imatinib resistance in gastrointestinal stromal tumors

Gastrointestinal stromal tumor （GIST） is one of the most common mesenchymal tumors of the gastrointestinal tract. Though imatinib improves the outcome, drug resistance remains the major problem for extending patient survival. Genetic mutation of the drug targets is the known mechanism for imatinib resistance. However, it cannot explain all of the phenomena of imatinib resistance, and numerous additional mechanisms have been proposed to account for imatinib resistance in various model systems. In this study, we applied the SYBR-green quantitative polymerase chain reaction-based array approach to screen the differentially expressed miRNAs between primary GIST patients and imatinib-resistant patients. The selected candidate miRNAs were validated in a cohort of 12 GIST patients. We found that low expression of miR-320a was correlated with short time to imatinib resistance, and proposed the potential mechanism ofmiR-320a for imatinib resistance.     

Comparative validation of c-kit exon 11 mutation analysis on cytology samples and corresponding surgical resections of gastrointestinal stromal tumours

Objective:  Studies have shown that c-kit mutation analysis of gastrointestinal stromal tumours (GISTs) obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) can be routinely performed. We validated c-kit exon 11 mutational analysis on cell block material obtained from fine needle aspiration cytology (FNAC) for diagnostic purposes and compared it with the same analysis in formalin-fixed paraffin-embedded full sections of the corresponding resection specimens.
Methods:  c-kit mutation analysis was done on cell block material obtained from ten cases encountered in our department from 1999 to 2008 on which FNAC was attempted pre-operatively. The findings were compared with analysis on full paraffin section of the corresponding resected tumours in seven cases where patients opted for resection. c-kit exon 11 was examined via bidirectional nucleic acid sequencing.
Results:  Our results showed 100% concordance for the presence and type of exon 11 mutation in the resected and aspirated tumours in all seven cases. These mutations had diagnostic value when compared with other neoplasms that are part of the cytomorphological differential diagnosis, such as leiomyosarcoma or gastric adenocarcinomas.
Conclusion:  Molecular cytopathology is a powerful tool that can complement morphology and immunohistochemical assessment of cytological material in routine practice for the diagnosis and prognostication of GISTs. We briefly discuss the advantages and limitations of the fine needle method of obtaining tissue for the diagnosis and prognostication of GISTs, and its current therapeutic strategies.     

Compartmentalization of integrin alpha6beta4 signaling in lipid rafts

Integrin alpha6beta4 signaling proceeds through Src family kinase (SFK)-mediated phosphorylation of the cytoplasmic tail of beta4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, alpha6beta4 mediates assembly of hemidesmosomes. Here, we report that part of alpha6beta4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of beta4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of alpha6beta4 in lipid rafts, but does not affect alpha6beta4-mediated adhesion or assembly of hemidesmosomes. The fraction of alpha6beta4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective alpha6beta4 does not activate SFK signaling to extracellular signal-regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the alpha6beta4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis.     

Autophagy is involved in endogenous and NVP-AUY922-induced KIT degradation in gastrointestinal stromal tumors

Gastrointestinal stromal tumor (GIST) is a prototype of mutant KIT oncogene-driven tumor. Prolonged tyrosine kinase inhibitor (TKI) treatment may result in a resistant phenotype through acquired secondary KIT mutation. Heat shock protein 90 (HSP90AA1) is a chaperone protein responsible for protein maturation and stability, and KIT is a known client protein of HSP90AA1. Inhibition of HSP90AA1 has been shown to destabilize KIT protein by enhancing its degradation via the proteasome-dependent pathway. In this study, we demonstrated that NVP-AUY922 (AUY922), a new class of HSP90AA1 inhibitor, is effective in inhibiting the growth of GIST cells expressing mutant KIT protein, the imatinib-sensitive GIST882 and imatinib-resistant GIST48 cells. The growth inhibition was accompanied with a sustained reduction of both total and phosphorylated KIT proteins and the induction of apoptosis in both cell lines. Surprisingly, AUY922-induced KIT reduction could be partially reversed by pharmacological inhibition of either autophagy or proteasome degradation pathway. The blockade of autophagy alone led to the accumulation of the KIT protein, highlighting the role of autophagy in endogenous KIT turnover. The involvement of autophagy in endogenous and AUY922-induced KIT protein turnover was further confirmed by the colocalization of KIT with MAP1LC3B-, acridine orange- or SQSTM1-labeled autophagosome, and by the accumulation of KIT in GIST cells by silencing either BECN1 or ATG5 to disrupt autophagosome activity. Therefore, the results not only highlight the potential application of AUY922 for the treatment of KIT-expressing GISTs, but also provide the first evidence for the involvement of autophagy in endogenous and HSP90AA1 inhibitor-induced KIT degradation.     

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis

Cancer‐associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF‐mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia‐inducible factor (HIF)‐1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF‐1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co‐injected with tumour cells similarly prevents CAF‐induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.     

Critical amino acid residues of the alpha4 subunit for alpha4beta7 integrin function

A characteristic feature of integrin-ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the alpha and beta subunit of the alpha4 integrins, alpha4beta1 and alpha4beta7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular domain of the murine and human alpha4-subunit contains three conserved LDV motifs, designated LDV-1 to -3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for alpha4beta7 integrins. We present evidence that LDV-1 mutants (D489N) behave like alpha4 wt cells, but LDV-3 mutants (D811N) are impaired in alpha4beta7 integrin-triggered homotypic cell aggregation and in adhesion and spreading on alpha4 specific ligands. Further characterization of LDV-3 mutants revealed a defect in mAb-induced alpha4beta7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of alpha4beta7 integrin cell surface expression. Our results indicate: (i) that LDV-3, located proximal to the cell membrane, is important for alpha4beta7 integrin-triggered functions and for lateral clustering and (ii) that LDV-2 affects alpha4beta7 heterodimer stability.     

Intracellular modifiers of integrin alpha 6p production in aggressive prostate and breast cancer cell lines

Cancer metastasis is a multi-step process in which tumor cells gain the ability to invade beyond the primary tumor and colonize distant sites. The mechanisms regulating the metastatic process confer changes to cell adhesion receptors including the integrin family of receptors. Our group previously discovered that the α6 integrin (ITGA6/CD49f) is post translationally modified by urokinase plasminogen activator (uPA) and its receptor, urokinase plasminogen activator receptor (uPAR), to form the variant ITGA6p. This variant of ITGA6 is a cleaved form of the receptor that lacks the ligand-binding domain. Although it is established that the uPA/uPAR axis drives ITGA6 cleavage, the mechanisms regulating cleavage have not been defined. Intracellular integrin dependent “inside-out” signaling is a major regulator of integrin function and the uPA/uPAR axis. We hypothesized that intracellular signaling molecules play a role in formation of ITGA6p to promote cell migration during cancer metastasis. In order to test our hypothesis, DU145 and PC3B1 prostate cancer and MDA-MB-231 breast cancer cell lines were treated with small interfering RNA targeting actin and the intracellular signaling regulators focal adhesion kinase (FAK), integrin linked kinase (ILK), and paxillin. The results demonstrated that inhibition of actin, FAK, and ILK expression resulted in significantly increased uPAR expression and ITGA6p production. Inhibition of actin increased ITGA6p, although inhibition of paxillin did not affect ITGA6p formation. Taken together, these results suggest that FAK and ILK dependent “inside-out” signaling, and actin dynamics regulate extracellular production of ITGA6p and the aggressive phenotype.     

SKP2 regulates ZEB1 expression and stimulates eutopic endometrial stromal cell invasion and proliferation of adenomyosis

Though endometriosis is benign, however, it shares certain characteristics with cancers, such as the ability to invade and metastasize. Previous studies have demonstrated that S-phase kinase associated protein2 (SKP2) promotes invasion, tumorigenesis, and metastasis. However, its correlation with adenomyosis is unclear. Herein, we aimed to look into SKP2 expression patterns and regulatory effects on endometrial stromal cell (ESC) proliferation and invasion, and its internal mechanism in adenomyosis. Western blot, qRT-PCR, and immunochemistry were carried out for detecting SKP2 and ZEB1 expression in ESC of adenomyosis and adenomyosis endometrial tissue. The primary ESCs were identified using immunofluorescence. SKP2 knockdown was accomplished in vitro by transfecting a particular lentivirus vector. The colony formation and CCK-8 assays were carried out for assessing cell proliferation, while cell invasion potential was assessed using the transwell assay. Both SKP2 and ZEB1 were found to be significantly upregulated in adenomyosis endometrial tissue. Knockdown of SKP2 inhibited adenomyotic ESC invasion and proliferation. Further experiments showed that knocking out SKP2 reduced ZEB1 expression in adenomyotic ESCs. Our results showed that SKP2 could regulate ZEB1 expression, and increased SKP2 may play a role in the pathogenesis of adenomyosis and stimulating ESC proliferation and invasion.     

Spatial restriction of alpha4 integrin phosphorylation regulates lamellipodial stability and alpha4beta1-dependent cell migration

Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.