Cytokine profiling of docetaxel-resistant castration-resistant prostate cancer.

BACKGROUND: Docetaxel improves symptoms and survival in metastatic castration-resistant prostate cancer (CRPC). However, ∼50% of patients are chemoresistant. This study examined whether changes in cytokine levels predict for docetaxel resistance in vitro and in a clinical cohort. METHODS: PC3 cells or their docetaxel-resistant subline (PC3Rx) were co-cultured with U937 monocytes, with and without docetaxel treatment, and cytokine levels were measured. The circulating levels of 28 cytokines were measured pre-/post cycle 1 of docetaxel from 55 men with CRPC, and compared with prostate-specific antigen (PSA) response. RESULTS: PC3Rx-U937 co-culture expressed more cytokines, chiefly markers of alternative macrophage differentiation, compared with PC3-U937 co-culture. Docetaxel treatment enhanced cytokine production by PC3Rx-U937 co-culture, while reducing cytokine levels in PC3-U937. In patients, changes in the levels of seven circulating cytokines (macrophage inhibitory cytokine 1 (MIC1), interleukin (IL)-1ra, IL-1ß, IL-4, IL-6, IL-12 and IFNγ) after cycle 1 of docetaxel were associated with progressive disease (all P<0.05). The combination of changes in MIC1, IL-4 and IL-6 most strongly predicted PSA response (P=0.002). CONCLUSIONS: In vitro studies suggest docetaxel resistance is mediated, at least in part, by cytokines induced by the interaction between the docetaxel-resistant tumour cells and macrophages. Early changes in circulating cytokine levels were associated with docetaxel resistance in CRPC patients. When considered together, these data suggest a significant role for the inflammatory response and macrophages in the development of docetaxel resistance in CRPC.

Circulating microRNAs are associated with docetaxel chemotherapy outcome in castration-resistant prostate cancer.

BACKGROUND: Docetaxel is the first-line chemotherapy for castration-resistant prostate cancer (CRPC). However, response rates are ∼50% and determined quite late in the treatment schedule, thus non-responders are subjected to unnecessary toxicity. The potential of circulating microRNAs as early biomarkers of docetaxel response in CRPC patients was investigated in this study. METHODS: Global microRNA profiling was performed on docetaxel-resistant and sensitive cell lines to identify candidate circulating microRNA biomarkers. Custom Taqman Array MicroRNA cards were used to measure the levels of 46 candidate microRNAs in plasma/serum samples, collected before and after docetaxel treatment, from 97 CRPC patients. RESULTS: Fourteen microRNAs were associated with serum prostate-specific antigen (PSA) response or overall survival, according to Mann-Whitney U or log-rank tests. Non-responders to docetaxel and patients with shorter survival generally had high pre-docetaxel levels of miR-200 family members or decreased/unchanged post-docetaxel levels of miR-17 family members. Multivariate Cox regression with bootstrapping validation showed that pre-docetaxel miR-200b levels, post-docetaxel change in miR-20a levels, pre-docetaxel haemoglobin levels and visceral metastasis were independent predictors of overall survival when modelled together. CONCLUSIONS: Our study suggests that circulating microRNAs are potential early predictors of docetaxel chemotherapy outcome, and warrant further investigation in clinical trials.

Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors.

Grb2-associated binder 2 (Gab2) serves as a critical amplifier in the signaling network of Bcr-Abl, the driver of chronic myeloid leukemia (CML). Despite the success of tyrosine kinase inhibitors (TKIs) in CML treatment, TKI resistance, caused by mutations in Bcr-Abl or aberrant activity of its network partners, remains a clinical problem. Using inducible expression and knockdown systems, we analyzed the role of Gab2 in Bcr-Abl signaling in human CML cells, especially with respect to TKI sensitivity. We show for the first time that Gab2 signaling protects CML cells from various Bcr-Abl inhibitors (imatinib, nilotinib, dasatinib and GNF-2), whereas Gab2 knockdown or haploinsufficiency leads to increased TKI sensitivity. We dissected the underlying molecular mechanism using various Gab2 mutants and kinase inhibitors and identified the Shp2/Ras/ERK and the PI3K/AKT/mTOR axes as the two critical signaling pathways. Gab2-mediated TKI resistance was associated with persistent phosphorylation of Gab2 Y452, a PI3K recruitment site, and consistent with this finding, the protective effect of Gab2 was completely abolished by the combination of dasatinib with the dual PI3K/mTOR inhibitor NVP-BEZ235. The identification of Gab2 as a novel modulator of TKI sensitivity in CML suggests that Gab2 could be exploited as a biomarker and therapeutic target in TKI-resistant disease.

Functional characterization of cancer-associated Gab1 mutations.

Grb2-associated binder 1 (Gab1) is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the epidermal growth factor receptor (EGFR). Although the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this paper we describe the functional characterization of these Gab1 mutants. MCF-10A immortalized mammary epithelial cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared with wild-type Gab1 controls. Expression of Gab1 or the mutants promoted epidermal growth factor (EGF)-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. The two mutants also enhanced branching morphogenesis in a different mammary epithelial cell line, HC11. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but ;not Y83. Cellular stimulation with EGF or hepatocyte growth factor (HGF) led to a transient, or sustained, induction of T387 phosphorylation, respectively. As T387 corresponds in position to Gab2 T391, which suppresses Gab2 signaling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative-feedback regulation of Gab1. Interrogation of publically-available databases revealed additional cancer-associated mutations at, or in close proximity to, identified serine/threonine phosphorylation sites in other docking proteins. These data indicate that aberrant Gab1 signaling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.

Annexin A6 is a scaffold for PKCα to promote EGFR inactivation.

Protein kinase Cα (PKCα) can phosphorylate the epidermal growth factor receptor (EGFR) at threonine 654 (T654) to inhibit EGFR tyrosine phosphorylation (pY-EGFR) and the associated activation of downstream effectors. However, upregulation of PKCα in a large variety of cancers is not associated with EGFR inactivation, and factors determining the potential of PKCα to downregulate EGFR are yet unknown. Here, we show that ectopic expression of annexin A6 (AnxA6), a member of the Ca(2+) and phospholipid-binding annexins, strongly reduces pY-EGFR levels while augmenting EGFR T654 phosphorylation in EGFR overexpressing A431, head and neck and breast cancer cell lines. Reduced EGFR activation in AnxA6 expressing A431 cells is associated with reduced EGFR internalization and degradation. RNA interference (RNAi)-mediated PKCα knockdown in AnxA6 expressing A431 cells reduces T654-EGFR phosphorylation, but restores EGFR tyrosine phosphorylation, clonogenic growth and EGFR degradation. These findings correlate with AnxA6 interacting with EGFR, and elevated AnxA6 levels promoting PKCα membrane association and interaction with EGFR. Stable expression of the cytosolic N-terminal mutant AnxA6(1-175), which cannot promote PKCα membrane recruitment, does not increase T654-EGFR phosphorylation or the association of PKCα with EGFR. AnxA6 overexpression does not inhibit tyrosine phosphorylation of the T654A EGFR mutant, which cannot be phosphorylated by PKCα. Most strikingly, stable plasma membrane anchoring of AnxA6 is sufficient to recruit PKCα even in the absence of EGF or Ca(2+). In summary, AnxA6 is a new PKCα scaffold to promote PKCα-mediated EGFR inactivation through increased membrane targeting of PKCα and EGFR/PKCα complex formation.

The antiproliferative effects of progestins in T47D breast cancer cells are tempered by progestin induction of the ETS transcription factor Elf5.

Prolactin and progesterone act together to regulate mammary alveolar development, and both hormones have been implicated in breast cancer initiation and progression. Here we show that Elf5, a prolactin-induced ETS transcription factor that specifies the mammary secretory cell lineage, is also induced by progestins in breast cancer cells via a direct mechanism. To define the transcriptional response to progestin elicited via Elf5, we made an inducible Elf5 short hairpin-RNA knock-down model in T47D breast cancer cells and used it to prevent the progestin-induction of Elf5. Functional analysis of Affymetrix gene expression data using Gene Ontologies and Gene Set Enrichment Analysis showed enhancement of the progestin effects on cell cycle gene expression. Cell proliferation assays showed a more efficacious progestin-induced growth arrest when Elf5 was kept at baseline levels. These results showed that progestin induction of Elf5 expression tempered the antiproliferative effects of progestins in T47D cells, providing a further mechanistic link between prolactin and progestin in the regulation of mammary cell phenotype.

Annexin A6 inhibits Ras signalling in breast cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) is associated with enhanced activation of wild-type (hyperactive) Ras in breast cancer. Little is known about the regulation of Ras inactivation and GTPase-activating proteins (GAPs), such as p120GAP, in cells with hyperactive Ras. Recently, we showed that in EGFR-overexpressing A431 cells, which lack endogenous Annexin A6 (AnxA6), ectopic expression of AnxA6 stimulates membrane recruitment of p120GAP to modulate Ras signalling. We now demonstrate that, AnxA6 is downregulated in a number of EGFR-overexpressing and estrogen receptor (ER)-negative breast cancer cells. In these cells, AnxA6 overexpression promotes Ca(2+)- and EGF-inducible membrane targeting of p120GAP. In ER-negative MDA-MB-436 cells, overexpression of p120GAP, but not CAPRI or a p120GAP mutant lacking the AnxA6-binding domain inhibits Ras/MAPK activity. AnxA6 knockdown in MDA-MB-436 increases Ras activity and cell proliferation in anchorage-independent growth assays. Furthermore, AnxA6 co-immunoprecipitates with H-Ras in a Ca(2+)- and EGF-inducible manner and fluorescence resonance energy transfer (FRET) microscopy confirmed that AnxA6 is in close proximity of active (G12V), but not inactive (S17N) H-Ras. Thus, association of AnxA6 with H-Ras-containing protein complexes may contribute to regulate p120GAP/Ras assembly in EGFR-overexpressing and ER-negative breast cancer cells.

Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis.

The Gab2 docking protein is a target of several oncogenic protein tyrosine kinases and potentiates activation of the Ras/extracellular signal regulated kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways. Since Gab2 is phosphorylated by c-Src, and both proteins are overexpressed in breast cancers, we have determined the biological consequences of their co-expression in the immortalized human mammary epithelial cell line MCF-10A. While overexpression of c-Src did not affect acinar morphogenesis or growth factor dependence in three-dimensional culture, c-Src co-operated with Gab2 to promote epidermal growth factor (EGF)-independent acinar growth. In contrast, expression of v-Src or the activated mutant c-SrcY527F led to a spectrum of aberrant phenotypes ranging from spheroids with incomplete luminal clearance to highly disrupted, dispersed structures. Gab2 co-expression shifted the phenotypic distribution towards the dispersed phenotype, an effect not observed with a Gab2 mutant unable to bind the p85 subunit of PI3-kinase (Gab2Deltap85). In v-Src-expressing cells, Gab2, but not Gab2Deltap85, significantly decreased E-cadherin adhesive strength without altering its surface expression. Gab2 associated with E-cadherin in the presence and absence of v-Src, indicating that the ability of Gab2 to weaken the strength of cell-cell contacts may reflect enhanced activation of PI3-kinase at adherens junctions. Gab2 also increased migration and invasion of these cells in transwell assays, but these effects were p85-independent. Overall, these findings demonstrate a novel mechanism whereby Gab2 may promote metastatic spread and indicate that Gab2 may play several roles during breast cancer progression.

Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein.

The BRAF(V600E) mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf(wt)), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5'-triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf(V600E), although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf(V600E) was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf(wt) and B-Raf(V600E) and suggests that B-Raf(V600E) cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation.

The ras signaling pathway in mammary tumorigenesis and metastasis.

The Ras superfamily of GTPases act as important regulatory switches to co-ordinate extracellular stimuli with activation of intracellular signaling pathways and appropriate biological responses. The Ras branch of this superfamily includes H-, K- and N-Ras, which are commonly mutated in particular human cancers, but notably not in those of the breast. Instead, in breast cancer the signaling pathways involving these GTPases may be upregulated due to increased coupling to growth factor receptors or other tyrosine kinases commonly overexpressed in this disease, or increased expression of regulators, the Ras protein itself, or downstream effectors. Functional studies utilizing both in vitro and in vivo models demonstrate that Ras signaling can regulate a variety of endpoints relevant to breast cancer progression, including anchorage dependent and independent growth, tumorigenesis, steroid sensitivity and invasion. Finally, analysis of the processing and signaling mechanisms of the Ras superfamily has identified potential targets for therapeutic intervention.

Identification of a novel human tankyrase through its interaction with the adaptor protein Grb14.

Tankyrase is an ankyrin repeat-containing poly(ADP-ribose) polymerase originally isolated as a binding partner for the telomeric protein TRF1, but recently identified as a mitogen-activated protein kinase substrate implicated in regulation of Golgi vesicle trafficking. In this study, a novel human tankyrase, designated tankyrase 2, was isolated in a yeast two-hybrid screen as a binding partner for the Src homology 2 domain-containing adaptor protein Grb14. Tankyrase 2 is a 130-kDa protein, which lacks the N-terminal histidine/proline/serine-rich region of tankyrase, but contains a corresponding ankyrin repeat region, sterile alpha motif module, and poly(ADP-ribose) polymerase homology domain. The TANKYRASE 2 gene localizes to chromosome 10q23.2 and is widely expressed, with mRNA transcripts particularly abundant in skeletal muscle and placenta. Upon subcellular fractionation, both Grb14 and tankyrase 2 associate with the low density microsome fraction, and association of these proteins in vivo can be detected by co-immunoprecipitation analysis. Deletion analyses implicate the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10-19 of tankyrase 2 in mediating this interaction. This study supports a role for the tankyrases in cytoplasmic signal transduction pathways and suggests that vesicle trafficking may be involved in the subcellular localization or signaling function of Grb14.

Up-regulation of the protein tyrosine phosphatase SHP-1 in human breast cancer and correlation with GRB2 expression.

The protein tyrosine phosphatase SHP-1 is predominantly expressed in hemopoietic cell lineages, where its function is relatively well defined. However, its expression profile also extends to certain epithelial cell types. Furthermore, the negative regulatory role of this enzyme in hemopoietic cell signaling may not apply to other systems, where positive effects on particular tyrosine kinase signaling pathways have been described. Expression of SHP-1 was therefore investigated in human breast cancer cell lines and primary breast cancers. Differential expression of SHP-1 mRNA was observed among the 19 breast cancer cell lines examined, and in an analysis of 72 primary breast cancers, SHP-1 mRNA expression was increased 2- to 12-fold relative to normal breast epithelial cells in 58% of the samples. Interestingly, a subset of the cancers also over-expressed GRB2 mRNA by 2- to 7-fold, and a significant (p < 0.01) positive correlation was observed between SHP-1 and GRB2 mRNA expression. Since these proteins can bind to each other and regulate MEK/MAP kinase activation, their co-ordinate up-regulation may amplify tyrosine kinase signaling in breast cancer cells.

Tyrosine kinase signalling in breast cancer: modulation of tyrosine kinase signalling in human breast cancer through altered expression of signalling intermediates.

The past decade has seen the definition of key signalling pathways downstream of receptor tyrosine kinases (RTKs) in terms of their components and the protein-protein interactions that facilitate signal transduction. Given the strong evidence that links signalling by certain families of RTKs to the progression of breast cancer, it is not surprising that the expression profile of key downstream signalling intermediates in this disease has also come under scrutiny, particularly because some exhibit transforming potential or amplify mitogenic signalling pathways when they are overexpressed. Reflecting the diverse cellular processes regulated by RTKs, it is now clear that altered expression of such signalling proteins in breast cancer may influence not only cellular proliferation (eg Grb2) but also the invasive properties of the cancer cells (eg EMS1/cortactin).

Signaling pathways and structural domains required for phosphorylation of EMS1/cortactin.

The structural characteristics of EMS1 (human cortactin) suggest that it may link signaling events to reorganization of the actin cytoskeleton. Interestingly, the EMS1 gene is commonly amplified and overexpressed in several human cancers, which may alter their invasive or metastatic properties. An 80 to 85-kDa mobility shift of EMS1 correlates with an alteration in subcellular distribution and is likely to represent an important regulatory event. In HEK 293 cells, epidermal growth factor treatment or cell detachment induced this shift, and this was blocked by the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059. Furthermore, expression of a constitutively active form of MEK induced the shift, indicating that MEK activation was both sufficient and necessary for this modification. The epidermal growth factor-induced shift correlated with increased phosphorylation on serine and threonine residues of the same tryptic phosphopeptides detected under basal conditions. Deletion of the helical-proline-rich region of the protein blocked the mobility shift and EMS1 phosphorylation. In vitro kinase assays demonstrated that the extracellular signal-regulated kinases represent candidate kinases for this region, although other MEK-regulated enzymes must also participate. These data identify MEK as an important intermediate involved in EMS1 phosphorylation and highlight the helical-proline-rich region as a key regulatory domain.

The Grb7 family of signalling proteins.

The Grb7 family is a rapidly emerging group of Src homology (SH)2 domain-containing signalling proteins that currently contains three members, Grb7, 10 and 14. These proteins possess a conserved multidomain structure, including a central region exhibiting significant homology to the Caenorhabditis elegans protein Mig10. Differences in tissue expression and SH2 binding selectivity suggest that these adaptor proteins function in a tissue-specific manner to link specific receptor tyrosine kinases (RTKs) and other tyrosine phosphorylated proteins to as yet uncharacterised downstream effectors. Interestingly, Grb7 proteins exhibit differential expression amongst a variety of human cancers and cancer cell lines. Consequently, these proteins not only are likely to perform a fundamental signalling role, but may also modulate RTK signalling in cancer cells.

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival.

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.

Inhibition of the MAP kinase cascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cells.

Members of the erbB family of receptor tyrosine kinases are commonly overexpressed in human breast cancer. However, the relative contribution of particular signalling pathways activated downstream of these receptors to the mitogenic response of transformed breast epithelial cells remains poorly characterized. Administration of heregulin-beta2 (HRG), a ligand for erbB3 and erbB4, to growth arrested T-47D human breast cancer cells leads to activation of both the PI3-kinase and MAP kinase signalling pathways and potent stimulation of cell cycle progression. Specific inhibitors were used to assess the role of these pathways in HRG-induced mitogenesis and to identify underlying mechanisms in terms of regulation of gene expression. Treatment with the MEK inhibitor PD98059 led to a complete block of HRG-induced entry into S-phase, whilst administration of the PI3-kinase inhibitor wortmannin resulted in only modest inhibition. In addition, administration of PD98059 8 h after HRG was equipotent with simultaneous administration in inhibiting entry into S-phase. However, delaying addition for 14-16 h after HRG, when the cells were entering S-phase, was without effect. HRG stimulation led to sequential induction of c-myc, cyclin D1, cyclin E and cyclin A gene expression and hyperphosphorylation of the retinoblastoma protein pRB. p21 (WAF1/CIP1/SDI1) gene expression was rapidly induced by HRG, but significant changes in p27 (KIP1) protein levels were not detected. Preincubation with PD98059 blocked the HRG-dependent induction of cyclin D1 mRNA, p21 and c-Myc protein and pRB phosphorylation. These findings demonstrate that MEK activation is critical to HRG-induced S-phase entry in these cells whilst PI3-kinase plays a minor role. Moreover, these data are compatible with HRG-induced activation of MEK being critical for a mid-G1 transition point and implicate c-myc and cyclin D1 as key targets of the MAP kinase pathway involved in this response.