Colette Galet

IGFBP-3 is a Metastasis Suppression Gene in Prostate Cancer

The insulin-like growth factor binding protein IGFBP-3 is a proapoptotic and antiangiogenic protein in prostate cancer (CaP). Epidemiologic studies suggest that low IGFBP-3 is associated with greater risk of aggressive, metastatic prostate cancers, but in vivo functional data are lacking. Here we show that mice that are genetically deficient in IGFBP-3 exhibit weaker growth of primary prostate tumors but higher incidence of metastatic disease. Prostates in IGFBP-3 knockout mice (IGFBP-3KO mice) failed to undergo apoptosis after castration. Spontaneous prostate tumors did not develop in IGFBP-3KO mice, but splenic lymphomas occurred in 23% of female IGFBP-3KO mice by 80 weeks of age. To assess the effects of IGFBP-3 deficiency on prostate cancer development, we crossed IGFBP-3KO mice with a c-Myc-driven model of CaP that develops slow-growing, nonmetastatic tumors. By 24 weeks of age, well-differentiated prostate cancers were observed in all mice regardless of IGFBP-3 status. However, by 80 weeks of age IGFBP-3KO mice tended to exhibit larger prostate tumors than control mice. More strikingly, lung metastases were observed at this time in 55% of the IGFBP-3KO mice but none in the control animals. Cell lines established from IGFBP-3KO:Myc tumors displayed more aggressive phenotypes in proliferation, invasion, and colony formation assays, relative to control Myc tumor cell lines. In addition, Myc:IGFBP-3KO cells exhibited evidence of epithelial-mesenchymal transition. Our findings established a function for IGFBP-3 in suppressing metastasis in prostate cancer, and they also offered the first reported transgenic model of spontaneous metastatic prostate cancer for studies of this advanced stage of disease.

Phase II Prospective Randomized Trial of a Low-Fat Diet with Fish Oil Supplementation in Men Undergoing Radical Prostatectomy

Preclinical studies suggest lowering dietary fat and decreasing the ratio of omega-6 to omega-3 polyunsaturated fatty acids decreases the risk of prostate cancer development and progression. We conducted a phase II randomized trial to test the effect of decreasing dietary fat combined with decreasing the dietary omega-6:omega-3 ratio on biomarkers related to prostate cancer development and progression. Patients undergoing radical prostatectomy were randomly assigned to receive a low-fat diet with 5 grams of fish oil daily (dietary omega-6:omega-3 ratio of 2:1) or a control Western diet (omega-6:omega-3 ratio of 15:1) for four to six weeks prior to surgery. The primary endpoint was change in serum insulin-like growth factor I (IGF-1) between arms. Secondary endpoints were serum IGFBP-1, prostate prostaglandin E2 levels, omega-6:omega-3 fatty acid ratios, COX-2, and markers of proliferation and apoptosis. Fifty-five patients were randomized and 48 completed the trial. There was no treatment difference in the primary outcome. Positive secondary outcomes in the low-fat fish oil versus Western group were reduced benign and malignant prostate tissue omega-6:omega-3 ratios, reduced proliferation (Ki-67 index), and reduced proliferation in an ex vivo bioassay when patient sera was applied to prostate cancer cells in vitro. In summary, four to six weeks of a low-fat diet and fish oil capsules to achieve an omega-6:omega-3 fatty acid ratio of 2:1 had no effect on serum IGF-1 levels, though in secondary analyses, the intervention resulted in decreased prostate cancer proliferation and decreased prostate tissue omega-6:omega-3 ratios. These results support further studies evaluating reduction of dietary fat with fish oil supplementation on modulating prostate cancer biology. Cancer Prev Res; 4(12); 2062–71. ©2011 AACR.

Effects of calorie restriction and IGF-1 receptor blockade on the progression of 22Rv1 prostate cancer xenografts.

Calorie restriction (CR) inhibits prostate cancer progression, partially through modulation of the IGF axis. IGF-1 receptor (IGF-1R) blockade reduces prostate cancer xenograft growth. We hypothesized that combining calorie restriction with IGF-1R blockade would have an additive effect on prostate cancer growth. Severe combined immunodeficient mice were subcutaneously injected with 22Rv1 cells and randomized to: (1) Ad libitum feeding/intraperitoneal saline (Ad-lib); (2) Ad-lib/20 mg/kg twice weekly, intraperitoneal ganitumab [anti-IGF-1R antibody (Ad-lib/Ab)]; (3) 40% calorie restriction/intraperitoneal saline (CR); (4) CR/ intraperitoneal ganitumab, (CR/Ab). CR and ganitumab treatment were initiated one week after tumor injection. Euthanasia occurred 19 days post treatment. Results showed that CR alone decreased final tumor weight, plasma insulin and IGF-1 levels, and increased apoptosis. Ganitumab therapy alone reduced tumor growth but had no effect on final tumor weight. The combination therapy (CR/Ab) further decreased final tumor weight and proliferation, increased apoptosis in comparison to the Ad-lib group, and lowered plasma insulin levels relative to the Ad-lib and Ad-lib/Ab groups. Tumor AKT activation directly correlated with plasma IGF-1 levels. In conclusion, whereas ganitumab therapy modestly affected 22Rv1 tumor growth, combining IGF-1R blockade with calorie restriction resulted in a significant decrease in final tumor weight and improved metabolic profile.

Effect of a low-fat fish oil diet on proinflammatory eicosanoids and cell-cycle progression score in men undergoing radical prostatectomy.

We previously reported that a 4- to 6-week low-fat fish oil (LFFO) diet did not affect serum insulin-like growth factor (IGF)-1 levels (primary outcome) but resulted in lower omega-6 to omega-3 fatty acid ratios in prostate tissue and lower prostate cancer proliferation (Ki67) as compared with a Western diet. In this post hoc analysis, the effect of the LFFO intervention on serum pro-inflammatory eicosanoids, leukotriene B4 (LTB4) and 15-S-hydroxyeicosatetraenoic acid [15(S)-HETE], and the cell-cycle progression (CCP) score were investigated. Serum fatty acids and eicosanoids were measured by gas chromatography and ELISA. CCP score was determined by quantitative real-time reverse transcriptase PCR (RT-PCR). Associations between serum eicosanoids, Ki67, and CCP score were evaluated using partial correlation analyses. BLT1 (LTB4 receptor) expression was determined in prostate cancer cell lines and prostatectomy specimens. Serum omega-6 fatty acids and 15(S)-HETE levels were significantly reduced, and serum omega-3 levels were increased in the LFFO group relative to the Western diet group, whereas there was no change in LTB4 levels. The CCP score was significantly lower in the LFFO compared with the Western diet group. The 15(S)-HETE change correlated with tissue Ki67 (R = 0.48; P < 0.01) but not with CCP score. The LTB4 change correlated with the CCP score (r = 0.4; P = 0.02) but not with Ki67. The LTB4 receptor BLT1 was detected in prostate cancer cell lines and human prostate cancer specimens. In conclusion, an LFFO diet resulted in decreased 15(S)-HETE levels and lower CCP score relative to a Western diet. Further studies are warranted to determine whether the LFFO diet antiproliferative effects are mediated through the LTB4/BLT1 and 15(S)-HETE pathways. Cancer Prev Res; 7(1); 97–104. ©2013 AACR.

Effect of a Low-Fat Diet Combined with IGF-1 Receptor Blockade on 22Rv1 Prostate Cancer Xenografts

In preclinical models, both dietary fat reduction and insulin-like growth factor I receptor (IGF-1R) blockade individually inhibit prostate cancer xenograft growth. We hypothesized that a low-fat diet combined with IGF-1R blockade would cause additive inhibition of prostate cancer growth and offset possible untoward metabolic effects of IGF-1R blockade antibody therapy. Fifty severe combined immunodeficient mice were injected with 22Rv1 cells subcutaneously. Ten days postinjection, the animals were randomized to four groups: (i) high-fat diet + saline (HF); (ii) high-fat diet + IGF-1R blocking antibody, ganitumab (HF/Ab); (iii) low-fat diet + saline (LF); and (iv) low-fat diet + ganitumab (LF/Ab). After 19 days of treatment, the animals were euthanized, serum was collected, and tumors were weighed. Tumor Ki67, Akt and extracellular signal-regulated kinase (ERK) activation, serum insulin, IGF-I and TNF-α were measured. In vitro, ganitumab treatment inhibited growth and induced apoptosis in several prostate cancer cell lines. In vivo, tumor weights and volumes were unaffected by the different treatments. The LF/Ab therapy significantly reduced proliferation (Ki67) and ERK activation in tumors. The HF/Ab group had significantly higher serum insulin levels than the HF group. However, LF/Ab combination significantly reduced serum insulin back to normal levels as well as normalizing serum TNF-α level. Whereas the combination of low-fat diet and IGF-1R blockade did not have additive inhibitory effects on tumor weight, it led to reduced tumor cell proliferation and a reduction in serum insulin and TNF-α levels. Mol Cancer Ther; 11(7); 1539–46. ©2012 AACR.

Global Igfbp1 deletion does not affect prostate cancer development in a c-Myc transgenic mouse model

Circulating insulin-like growth factor binding protein 1 (IGFBP1) levels vary in response to nutritional status, and pre-clinical studies suggest that elevated IGFBP1 may be protective against the development and progression of prostate cancer. We hypothesized that global deletion of Igfbp1 would accelerate the development of prostate cancer in a c-Myc transgenic mouse model. To test our hypothesis, c-Myc transgenic mice (Myc/BP-1 wild-type (WT)) were crossed and interbred with the Igfbp1 knockout mice (Myc/BP-1 KO). The animals were placed on a high-protein diet at weaning, weighed every 2 weeks, and euthanized at 16 weeks of age. Prostate histopathology was assessed and proliferation status was determined by Ki-67 and proliferating cell nuclear antigen analyses. IGF-related serum biomarkers and body composition were measured. No significant difference in the incidence of prostate cancer was observed between the Myc/BP-1 KO and the Myc/BP-1 WT mice (65 and 80% respectively, P=0.48). Proliferation was significantly decreased by 71% in prostate tissue of Myc/BP-1 KO mice compared with Myc/BP-1 WT mice. Myc/BP-1 KO mice exhibited a significant 6.7% increase in body weight relative to the Myc/BP-1 WT mice that was attributed to an increase in fat mass. Fasting insulin levels were higher in the Myc/BP-1 KO mice without any difference between the groups in fasting glucose concentrations. Thus, contrary to our hypothesis, global deletion of Igfbp1 in a c-Myc transgenic mouse model did not accelerate the development of prostate cancer. Global Igfbp1 deletion did result in a significant increase in body weight and body fat mass. Further studies are required to understand the underlying mechanisms for these metabolic effects.

Reply to “Phase II prospective randomized trial of weight loss prior to radical prostatectomy”

BackgroundObesity is associated with poorly differentiated and advanced prostate cancer and increased mortality. In preclinical models, caloric restriction delays prostate cancer progression and prolongs survival. We sought to determine if weight loss (WL) in men with prostate cancer prior to radical prostatectomy affects tumor apoptosis and proliferation, and if WL effects other metabolic biomarkers.MethodsIn this Phase II prospective trial, overweight and obese men scheduled for radical prostatectomy were randomized to a 5–8 week WL program consisting of standard structured energy-restricted meal plans (1200–1500 Kcal/day) and physical activity or to a control group. The primary endpoint was apoptotic index in the radical prostatectomy malignant epithelium. Secondary endpoints were proliferation (Ki67) in the radical prostatectomy tissue, body weight, body mass index (BMI), waist to hip ratio, body composition, and serum PSA, insulin, triglyceride, cholesterol, testosterone, estradiol, leptin, adiponectin, interleukin 6, interleukin 8, insulin-like growth factor 1, and IGF binding protein 1.ResultsIn total 23 patients were randomized to the WL intervention and 21 patients to the control group. Subjects in the intervention group had significantly more weight loss (WL:−3.7 ± 0.5 kg; Control:−1.6 ± 0.5 kg; p = 0.007) than the control group and total fat mass was significantly reduced (WL:−2.1 ± 0.4; Control: 0.1 ± 0.3; p = 0.015). There was no significant difference in apoptotic or proliferation index between the groups. Among the other biomarkers, triglyceride, and insulin levels were significantly decreased in the WL compared with the control group.ConclusionsIn summary, this short-term WL program prior to radical prostatectomy resulted in significantly more WL in the intervention vs. the control group and was accompanied by significant reductions in body fat mass, circulating triglycerides, and insulin. However, no significant changes were observed in malignant epithelium apoptosis or proliferation. Future studies should consider a longer term or more intensive weight loss intervention.

Keratin 13 Is Enriched in Prostate Tubule-Initiating Cells and May Identify Primary Prostate Tumors that Metastasize to the Bone

Background Benign human prostate tubule-initiating cells (TIC) and aggressive prostate cancer display common traits, including tolerance of low androgen levels, resistance to apoptosis, and microenvironment interactions that drive epithelial budding and outgrowth. TIC can be distinguished from epithelial and stromal cells that comprise prostate tissue via cell sorting based upon Epcam, CD44, and CD49f antigenic profiles. Fetal prostate epithelial cells (FC) possess a similar antigenic profile to adult TIC and are capable of inducing tubule formation. To identify the TIC niche in human prostate tissue, differential keratin (KRT) expression was evaluated. Results Gene expression data generated from Affymetrix Gene Chip human U133 Plus 2.0 array of sorted adult and fetal epithelial cells revealed KRT13 to be significantly enriched in FC and TIC compared to basal cells (BC) and luminal cells (LC) (p<0.001). Enriched KRT13 expression was confirmed by RT-PCR and cytospin immunostaining. Immunohistochemical analysis of KRT13 expression revealed rare KRT13+ epithelia throughout prostatic ducts/acini in adult tissue specimens and differentiated tubules in 24-week recombinant grafts, In contrast, abundant KRT13 expression was observed in developing ducts/acini in fetal prostate and cord-like structures composing 8-week recombinant grafts. Immunostaining of a prostate tissue microarray revealed KRT13+ tumor foci in approximately 9% of cases, and this subset displayed significantly shorter time to recurrence (p = 0.031), metastases (p = 0.032), and decreased overall survival (p = 0.004). Diagnostic prostate needle biopsies (PNBX) from untreated patients with concurrent bone metastases (clinical stage M1) displayed KRT13+ tumor foci, as did bone metastatic foci. Conclusions The expression profile of KRT13 in benign fetal and adult prostate tissue and in recombinant grafts, as well as the frequency of KRT13 expression in primary and metastatic prostate cancer indicates that it may be a marker of a stem/progenitor-like cell state that is co-opted in aggressive tumor cells. KRT13 is enriched in benign stem-like cells that display androgen-resistance, apoptosis-resistance, and branching morphogenesis properties. Collectively our data demonstrate that KRT13 expression is associated with poor prognosis at multiple stages of disease progression and may represent an important biomarker of adverse outcome in patients with prostate cancer.

Dietary Omega-6 and Omega-3 Fatty Acids and Prostate Cancer – Response

We truly appreciate the comments from Drs. Azrad and Demark-Wahnefried with regard to the potential for the proliferation bioassay reported in our article to potentially act as a surrogate biomarker for proliferation in the prostate cancer tissue. We would first like to clarify that there are 2