Impact of carbohydrate restriction in the context of obesity on prostate tumor growth in the Hi-Myc transgenic mouse model.

BACKGROUND: Previously, we showed that carbohydrate restriction with calorie restriction slowed tumor growth in xenograft mouse prostate cancer models. Herein, we examined the impact of carbohydrate restriction without calorie restriction on tumor development within the context of diet-induced obesity in the Hi-Myc transgenic mouse model of prostate cancer. METHODS: Mice were randomized at 5 weeks of age to ad libitum western diet (WD; 40% fat, 42% carbohydrate; n=39) or ad libitum no carbohydrate ketogenic diet (NCKD; 82% fat, 1% carbohydrate; n=44). At age 3 or 6 months, mice were killed, prostates weighed and prostate histology, proliferation, apoptosis and macrophage infiltration evaluated by hematoxylin and eosin, Ki67, TUNEL and F4/80 staining, respectively. Body composition was assessed by DEXA, serum cytokines measured using multiplex, and Akt/mTOR signaling assessed by Western. RESULTS: Caloric intake was higher in the NCKD group, resulting in elevated body weights at 6 months of age, relative to the WD group (45 g vs 38g; P=0.008). Despite elevated body weights, serum monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1α levels were lower in NCKD versus WD mice (P=0.046 and P=0.118, respectively), and macrophage infiltration was reduced in prostates of NCKD versus WD mice (P=0.028). Relative Akt phosphorylation and phospho-S6 ribosomal protein levels were reduced in prostates of NCKD versus WD mice. However, while mice randomized to NCKD had smaller prostates after adjustment for body weight at 3 and 6 months (P=0.004 and P=0.002, respectively), NCKD mice had higher rates of adenocarcinoma at 6 months compared to WD mice (100 vs 80%, P=0.04). CONCLUSIONS: Despite higher caloric intake and elevated body weights, carbohydrate restriction lowered serum MCP-1 levels, reduced prostate macrophage infiltration, reduced prostate weight, but failed to slow adenocarcinoma development. Together, these data suggest that although carbohydrate restriction within the context of obesity may reduce obesity-associated systemic inflammation and perhaps slow tumor growth, it is not sufficient to counteract obesity-associated tumor development.

PTEN loss and chromosome 8 alterations in Gleason grade 3 prostate cancer cores predicts the presence of un-sampled grade 4 tumor: implications for active surveillance.

Men who enter active surveillance because their biopsy exhibits only Gleason grade 3 (G3) frequently have higher grade tumor missed by biopsy. Thus, biomarkers are needed that, when measured on G3 tissue, can predict the presence of higher grade tumor in the whole prostate. We evaluated whether PTEN loss, chromosome 8q gain (MYC) and/or 8p loss (LPL) measured only on G3 cores is associated with un-sampled G4 tumor. A tissue microarray was constructed of prostatectomy tissue from patients whose prostates exhibited only Gleason score 3+3, only 3+4 or only 4+3 tumor (n=50 per group). Cores sampled only from areas of G3 were evaluated for PTEN loss by immunohistochemistry, and PTEN deletion, LPL/8p loss and MYC/8q gain by fluorescence in situ hybridization. Biomarker results were compared between Gleason score 6 vs 7 tumors using conditional logistic regression. PTEN protein loss, odds ratio=4.99, P=0.033; MYC/8q gain, odds ratio=5.36, P=0.010; and LPL/8p loss, odds ratio=3.96, P=0.003 were significantly more common in G3 cores derived from Gleason 7 vs Gleason 6 tumors. PTEN gene deletion was not statistically significant. Associations were stronger comparing Gleason 4+3 vs 6 than for Gleason 3+4 vs 6. MYC/8q gain, LPL/8p loss and PTEN protein loss measured in G3 tissue microarray cores strongly differentiate whether the core comes from a Gleason 6 or Gleason 7 tumor. If validated to predict upgrading from G3 biopsy to prostatectomy these biomarkers could reduce the likelihood of enrolling high-risk men and facilitate safe patient selection for active surveillance.

Normal and malignant prostate epithelial cells differ in their response to hepatocyte growth factor/scatter factor.

Hepatocyte growth factor/scatter factor (HGF/SF) promotes the proliferation, differentiation, motility, and invasion of epithelial cells by binding to its cell surface receptor, the Met tyrosine kinase. In the prostate, Met is expressed predominantly by prostate epithelial cells (PrEC), whereas HGF/SF is synthesized by prostate stromal cells (PrSC). Met is also expressed in localized and metastatic prostate cancers. Our results show that PrECs in in vitro culture maintain expression of Met at a level comparable to DU145 cancer cell expression. HGF/SF secreted by PrSC stimulates tyrosine phosphorylation of the Met receptor. In normal PrEC, HGF/SF causes growth inhibition, sustained phosphorylation of mitogen-activated protein kinase, and increased CK18 expression consistent with cell differentiation. In contrast, HGF/SF significantly stimulates the proliferation of DU145 prostate cancer cells. HGF/SF in the conditioned medium of PrSC specifically induces migration of both normal and malignant prostate epithelial cells through MatriGel-coated Transwell filters. HGF/SF depletion reduces cell migration by approximately 50%. The response of PrEC is specific for HGF/SF since the other growth factors tested do not significantly affect growth or migration of PrECs. These results support the in vivo importance of the prostate stroma and specifically of HGF/SF as a unique stromal derived factor in the development and progression of prostate cancer.

BCL-2 and p53 expression in clinically localized prostate cancer predicts response to external beam radiotherapy.

PURPOSE: Clinicians have long been hampered by the inability to distinguish patients with localized prostate cancer who will and will not respond to radiotherapy. In a significant proportion of patients therapy fails as determined by increasing posttreatment serum prostate specific antigen (PSA). We evaluated the expression of 2 key regulators of apoptosis, bcl-2 and p53, relative to treatment outcomes in patients who received external beam radiotherapy for clinically organ confined carcinoma of the prostate. MATERIALS AND METHODS: Immunohistochemical staining for bcl-2 and p53 on pretreatment needle biopsies was performed in 54 patients who were treated with radiotherapy for localized prostate cancer. Expression was scored using strict criteria. Nadir PSA less than 1 ng./ml. after therapy was considered a successful treatment response. RESULTS: There was a predominance of stage T1c cancer (74%) with a mean Gleason score of 6.9 and an average pretreatment PSA of 25.3 ng./ml. Overall 54% of the patients did not have a nadir PSA of less than 1 ng./ml. Of the bcl-2 positive cases therapy ultimately failed in 85%. Similarly 88% of the patients with p53 positive biopsies had treatment failure and in all with bcl-2 as well as p53 expression radiotherapy failed. Expression of bcl-2 and p53 was an independent prognostic variable for treatment failure with odds ratios (95% confidence interval) of 7.3 and 10.8, respectively. CONCLUSIONS: Expression of bcl-2 and p53 was associated with treatment failure after external beam radiation therapy. These findings suggest that bcl-2 and p53 expression in pretreatment biopsies may be helpful for predicting response to definitive radiotherapy.

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

Physiological signals and oncogenesis mediated through Crk family adapter proteins.

The viral Crk oncogene (v-Crk) is known to induce sarcomas in chicken and its cellular homologs c-Crk I, c-Crk II, and Crk-like (CRKL) have been implicated in many signal transduction events. These include cell differentiation, cell migration, and the induced nonresponsiveness of T-cells to stimulation of the T-cell receptor (TCR), a state known as anergy. CRKL is also the most prominent substrate of the Bcr-Abl oncoprotein which causes human chronic myelogenous leukemias (CML). The modular composition of the Crk family adapters which largely consist of Src homology (SH2 and SH3) domains has prompted an intensive search for physiological and pathological upstream and downstream signalling partners which selectively bind to these adapters. Upstream proteins include various receptors and large multisite docking proteins, while several protein kinases and guanine nucleotide release proteins (GNRPs) have been suggested to function downstream of c-Crk and CRKL. Most Crk/CRKL SH2- and SH3-binding proteins contain several docking sites with considerable sequence similarity. Thus the binding requirements of Crk/CRKL SH2 and SH3 domains are now well defined, providing a basis for the design of small inhibitory molecules to block the function of these adapter proteins. The enzymatic cascades activated through Crk family adapters are only partially known, but stress kinases (SAPKs/JNKs) and the GTPase Rap1, as well as the B-Raf isoform of the Raf protein kinases, are affected in some systems. Several yet unidentified, highly selective Crk interacting proteins detectable in specific cell types remain to be studied. More detailed analyses of the enzymatic activities triggered through Crk-type adapters will also be crucial to fully define the signalling pathways controlled by this protein family.

SH2 and SH3-containing adaptor proteins: redundant or independent mediators of intracellular signal transduction.

Molecules which contain Src Homology 2 (SH2) and SH3 domains provide one of the principal ways by which signals are transduced in cells using protein-protein interactions between proline-rich motifs and SH3 domains and induced interactions between phosphotyrosine residues and SH2 domains. The simplest of SH2/SH3-containing proteins are the Crk, Grb2 and Nck adaptor proteins which contain SH2 and SH3 domains but no intrinsic catalytic activity. Whereas Grb2 connects activated receptor tyrosine kinases with Sos and activates p21ras, recent evidence suggests that this may not be the major mechanism by which Crk and Nck signal to downstream effectors. Identification of novel binding partners for Crk, Grb2 and Nck indicate that these adaptor proteins control distinct aspects of tyrosine kinase signalling.

The SH3 domain of Crk binds specifically to a conserved proline-rich motif in Eps15 and Eps15R.

The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are thought to transduce signals from tyrosine kinases to downstream effectors. In order to understand the specificity and effector function of the SH3 domain of Crk, we screened an expression library for binding proteins. We isolated Eps15, a substrate of the epidermal growth factor receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequence homology to the carboxyl-terminal domain of Eps15. Antibodies raised against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa. Eps15 and Eps15R bound specifically to the amino-terminal SH3 domain of Crk and coprecipitated equivalently with both c-Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 and Eps15R featured several proline-rich regions as putative binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified one proline-rich motif which accounts for their interaction with the Crk SH3 domain. Each binding motif contains the sequence P-X-L-P-X-K, an amino acid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro-translated Eps15 only in the presence of in vitro-translated v-Crk. Crk might therefore be involved in Eps15-mediated signal transduction through the EGFR.

Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins.

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a Kd of 1.89 +/- 0.06 microM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c-Crk and v-Crk further suggested that K10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.

Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk.

The widely expressed cellular Crk protein has the domain structure SH2-SH3-SH3. We have previously demonstrated that the more N-terminal SH3 domain of Crk (CrkSH3(N)) specifically binds several cytoplasmic proteins. A cDNA encoding one of these proteins was isolated and found to have two different splice forms. The sequence is virtually identical to C3G, a guanine-nucleotide exchange factor. The center region of the 145-155-kDa protein contains four similar proline-rich sequences which are capable of binding individually to the SH3(N) domains of c-Crk and v-Crk. Comparison of these sequences in C3G to proline-rich sequences in other Crk-binding proteins suggests that positively charged amino acids following the prolines play an important role in the binding to the CrkSH3(N) domain. The endogenous C3G could be coprecipitated with Crk from cell lysates of cells expressing high levels of c-Crk or v-Crk, suggesting high binding affinity and a possible interaction in vivo. Unlike many other SH3-binding proteins which interact with multiple SH3 domains, C3G from cell lysates binds preferentially to the CrkSH3(N) domain. This unique binding specificity supports the idea that C3G plays an important role in Crk signaling pathways.

Matrix plasminogen activator inhibitor. Modulation of the extracellular proteolytic environment.

We have previously demonstrated that plasminogen activator inhibitor (PAI-1) is associated with the extracellular matrix of cultured bovine smooth muscle cells (Knudsen, B.S., Harpel, P.C., Nachman, R.L. (1987) J. Clin. Invest. 80, 1082-1089). In this report we describe the physiologic role of PAI-1 during the interaction of the tissue plasminogen activator (t-PA) secreting Bowes human melanoma cell line with endothelial extracellular matrices. In addition we have characterized the t-PA.PAI complexes formed during this interaction in the presence and absence of plasminogen. In the absence of plasminogen, a 104-kDa complex between Bowes t-PA and PAI-1 appears in the supernatant. In the presence of plasminogen, PAI initially prevents plasmin formation on the matrix and protects the matrix from degradation by plasmin. The 104-kDa t-PA.PAI complex is degraded into a 68 and a 47-kDa complex by small amounts of plasmin generated from secreted Bowes t-PA and plasminogen. Analysis of these complexes revealed that t-PA is rapidly cleaved by plasmin within the complex whereas complexed PAI-1 is not further degraded. Matrix-associated PAI-1 may play an important role in the protection of extracellular matrices from remodeling and degradation by cellular t-PA and plasminogen.

Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells.

The extracellular matrix secreted by cultured bovine smooth muscle cells (BSMC) contains an endothelial type plasminogen activator (PA) inhibitor. When PA is incubated with the matrix, a high molecular weight complex containing a truncated PA inhibitor is released into the supernatant. The inhibitor also dissociates from the matrix by treatment with glycine, pH 2.7, in its intact, functionally active, 45-kD form, whereas treatment of the matrix with thrombin results in the release of a cleaved, inactive, 41 kD PA inhibitor. Bowes melanoma cells but not smooth muscle cells cultured on BSMC matrices decrease available matrix associated PA inhibitor. PA inhibitor incorporated into the extracellular matrix may serve an important role in the regulation of plasminogen activator mediated matrix degradation.

Binding of plasminogen to extracellular matrix.

We have previously demonstrated that plasminogen immobilized on various surfaces forms a substrate for efficient conversion to plasmin by tissue plasminogen activator (t-PA) (Silverstein, R. L., Nachman, R. L., Leung, L. L. K., and Harpel, R. C. (1985) J. Biol. Chem. 260, 10346-10352). We now report the binding of human plasminogen to the extracellular matrix synthesized in vitro by cultured endothelial cell monolayers. The binding was specific, saturable at plasma plasminogen concentrations, reversible, and lysine-binding site-dependent. Functional studies demonstrated that matrix immobilized plasminogen was a much better substrate for t-PA than was fluid phase plasminogen as shown by a 100-fold decrease in Km. Activation of plasminogen by t-PA and urokinase on the matrix was equally efficient. The plasmin generated on the matrix, in marked contrast to fluid phase, was protected from its fast-acting inhibitor, alpha 2-plasmin inhibitor. Matrix-associated plasmin converted bound Glu- into Lys-plasminogen, which in turn is more rapidly activated to plasmin by t-PA. The extracellular matrix not only binds and localizes plasminogen but also improves plasminogen activation kinetics and prolongs plasmin activity in the subendothelial microenvironment.