Leucine can modulate skeletal muscle metabolism by enhancing protein synthesis and decreasing proteolysis. In this study, we investigated the effects of leucine on the ubiquitin–proteasome system in skeletal muscle of pregnant tumour-bearing rats fed a leucine-rich diet. Pregnant Wistar rats were distributed into three groups that were fed a semi-purified control diet (C, control; W, Walker tumour-bearing; P, pair-fed) and three other groups of pregnant rats fed a semi-purified leucine-rich diet (L, leucine; WL, Walker tumour-bearing; PL, pair-fed). The tumour-bearing rats were injected subcutaneously with a suspension of Walker 256 tumour cells. Protein synthesis and degradation were measured in gastrocnemius muscle; the total protein content and tissue chymotrypsin-like and alkaline phosphatase enzyme activities were also determined. Muscle protein extracts were run on SDS-PAGE to assess the expression of the myosin heavy chain (MHC), 20S α proteasome subunit, 19S MSSI ATPase regulator subunit and 11S α subunit. Although tumour growth decreased the incorporation of [3H]-Phe, the concomitant feeding of a leucine-rich diet increased the rate of protein synthesis. Muscle proteolysis in both tumour-bearing groups was increased more than in the respective control groups. Conversely, the leucine-rich diet caused less protein breakdown in the WL group than in the W group. Only the W group showed a significant reduction (71%) in the myosin content. In WL rats, the 20S proteasome content (32 kDa band) was reduced, while the expression of the 19S subunit was 3-fold less than in the W group and the 11S proteasome subunit reduced, to around 32% less than in the W group. These findings clearly indicate that leucine can stimulate protein synthesis and inhibit protein breakdown in pregnant rats, probably by modulating the activation of the ubiquitin–proteasome system during tumour growth.
G Ventrucci, M A R Mello and M C C Gomes-Marcondes
Manali Mody, Nachiket Dharker, Mark Bloomston, Pei-Shan Wang, Fu-Sheng Chou, Theodore S Glickman, Timothy McCaffrey, Zhaoqing Yang, Anne Pumfery, Daniel Lee, Matthew D Ringel and Joseph J Pinzone
Peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear hormone superfamily and has multiple endogenous and pharmacological ligands, including 15-deoxy-Δ 12,14-prostaglandin J2 and two thiazolidinediones (TZD), rosiglitazone and pioglitazone, which are used clinically to treat type-2 diabetes mellitus. PPARγ agonists regulate development, cellular growth and metabolism in various tissues and have been documented to decrease cellular proliferation and to induce apoptosis of various tumour phenotypes, including breast cancer. However, the full spectrum of anti-tumour effects occurs only at suprapharmacological doses. In this study, we investigated the mechanism of rosiglitazone-induced anti-tumour effects of MDA-MB-231 human breast cancer cells, and used that information to predict rosiglitazone-induced sensitization of breast cancer cells to the effects of other compounds. We first confirmed that 100 μM rosiglitazone, but not lower doses, decreases MDA-MB-231 cell viability in vitro. We then used microarray gene expression analysis to determine early rosiglitazone-induced gene expression changes after 4-h exposure, which included 1298 genes that we grouped into functional categories. We selectively confirmed rosiglitazone-mediated effects on expression of key regulators of breast cancer proliferation and apoptosis, including p53, p21 and Bax. Finally, we used this information to predict that rosiglitazone would sensitize MDA-MB-231 cells to the anti-tumour effects of CH11, which trimerizes Fas, as well as tumour necrosis factor-α. Moreover, we used the confirmed array data to predict cooperative activity of rosiglitazone and R-roscovitine (CYC202), an inhibitor of multiple cyclin-dependent kinases. We conclude that microarray analysis can determine early TZD-modulated changes in gene expression that help to predict effective in vitro drug combinations.
Mark A White, Efrosini Tsouko, Chenchu Lin, Kimal Rajapakshe, Jeffrey M Spencer, Sandi R Wilkenfeld, Sheiva S Vakili, Thomas L Pulliam, Dominik Awad, Fotis Nikolos, Rajasekhara Reddy Katreddy, Benny Abraham Kaipparettu, Arun Sreekumar, Xiaoliu Zhang, Edwin Cheung, Cristian Coarfa and Daniel E Frigo
Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression of SLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated with SLC2A12 expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5′-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increased TBC1D4 expression. Correspondingly, expression of TBC1D4 correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.
D J Liao and R B Dickson
Ever since Bishop and his co-workers discovered the c-myc gene in the late 1970s (Bishop 1982), voluminous literature has documented its central role in proliferation and malignant transformation of human and animal cells (Amati et al. 1998, Bouchard et al. 1998, Dang et al. 1999). Most, if not all, types of human malignancy have been reported to have amplification and/or overexpression of this gene, although the frequency of these alterations varies greatly among different reports (Nesbit et al. 1999). In 1992, researchers started to realize that aberrant expression of c-myc could cause apoptosis (Evan et al. 1992, Shi et al. 1992), although the phenomenon had actually been observed much earlier (Wurm et al. 1986). Studies in recent years have further shown that the c-myc gene regulates growth, both in the sense of cell size and in the context of tissue differentiation (Gandarillas & Watt 1997, Iritani & Eisenman 1999, Johnston et al. 1999, Schmidt 1999, Schuhmacher et al. 1999). Thus, it is now known that the c-myc gene participates in most aspects of cellular function, including replication, growth, metabolism, differentiation, and apoptosis (Packham & Cleveland 1995, Hoffman & Liebermann 1998, Dang 1999, Dang et al. 1999, Elend & Eilers 1999, Prendergast 1999). How the c-Myc protein may be specifically directed to perform one, but not the others, of these functions is still obscure, despite the fact that the relevant literature has been accumulating at a fast pace in the past two decades. This review focuses on the profound roles of c-Myc in breast cancer and in the actions of the hormones that are eitologically related to breast cancer.
Jingqi Fu, Hongzhi Zheng, Qi Cui, Chengjie Chen, Simeng Bao, Jing Sun, Lu Li, Bei Yang, Huihui Wang, Yongyong Hou, Yuanyuan Xu, Yuanhong Xu, Qiang Zhang and Jingbo Pi
The transcription factor nuclear factor erythroid 2-like 1 (NFE2L1 or NRF1) is involved in various critical cell processes such as maintenance of ubiquitin-proteasome system and regulation of the cellular antioxidant response. We previously determined that pancreatic β-cell-specific Nfe2l1-knockout mice had hyperinsulinemia and that silencing of Nfe2l1 in mouse islets or MIN6 insulinoma β-cells induced elevated basal insulin release and altered glucose metabolism. Hypoglycemia is a major issue with aggressive insulinomas, although a role of NFE2L1 in this pathology is not defined. In the present work, we studied the tumorigenicity of Nfe2l1-deficient insulinoma MIN6 cells (Nfe2l1-KD) and sensitivity to chemotherapy. Nfe2l1-KD cells grew faster and were more aggressive than Scramble cells in vitro. In a mouse allograft transplantation model, insulinomas arising from Nfe2l1-KD cells were more aggressive and chemoresistant. The conclusion was amplified using streptozotocin (STZ) administration in an allograft transplantation model in diabetic Akita background mice. Furthermore, Nfe2l1-KD cells were resistant to damage by the chemotherapeutic drugs STZ and 5-fluorouracil, which was linked to binding of hexokinase 1 with mitochondria, enhanced mitochondrial membrane potential and closed mitochondrial potential transition pore. Overall, both in vitro and in vivo data from Nfe2l1-KD insulinoma cells provided evidence of a previously un-appreciated action of NFE2L1 in suppression of tumorigenesis. Nfe2l1 silencing desensitizes insulinoma cells and derived tumors to chemotherapeutic-induced damage, likely via metabolic reprograming. These data indicate that NFE2L1 could potentially play an important role in the carcinogenic process and impact chemosensitivity, at least within a subset of pancreatic endocrine tumors.
Lei Quan, Chi-Chen Hong, Gary Zirpoli, Michelle R Roberts, Thaer Khoury, Lara E Sucheston-Campbell, Dana H Bovbjerg, Lina Jandorf, Karen Pawlish, Gregory Ciupak, Warren Davis, Elisa V Bandera, Christine B Ambrosone and Song Yao
It has been observed previously that compared with women of European ancestry (EA), those of African ancestry (AA) are more likely to develop estrogen receptor (ER)-negative breast cancer, although the mechanisms have not been elucidated. We tested the associations between breast cancer risk and a targeted set of 20 genes known to be involved in estrogen synthesis, metabolism, and response and potential gene–environment interactions using data and samples from 1307 EA (658 cases) and 1365 AA (621 cases) participants from the Women’s Circle of Health Study (WCHS). Multivariable logistic regression found evidence of associations with single-nucleotide polymorphisms (SNPs) in the ESR1 gene in EA women (rs1801132, odds ratio (OR)=1.47, 95% CI=1.20–1.80, P=0.0002; rs2046210, OR=1.24, 95% CI=1.04–1.47, P=0.02; and rs3020314, OR=1.43, 95% CI=1.19–1.70, P=0.00009), but not in AA women. The only other gene associated with breast cancer risk was CYP1A2 in AA women (rs2470893, OR=1.42, 95% CI=1.00–2.02, P=0.05), but not in EA women. When stratified by ER status, ESR1 rs1801132, rs2046210, and rs3020314 showed stronger associations in ER-positive than in ER-negative breast cancer in only EA women. Associations with the ESR1 SNPs in EA women also appeared to be stronger with longer endogenous estrogen exposure or hormonal replacement therapy use. Our results indicate that there may be differential genetic influences on breast cancer risk in EA compared with AA women and that these differences may be modified by tumor subtype and estrogen exposures. Future studies with a larger sample size may determine the full contribution of estrogen-related genes to racial/ethnic differences in breast cancer.
Isabelle Laverdière, Christine Flageole, Étienne Audet-Walsh, Patrick Caron, Yves Fradet, Louis Lacombe, Éric Lévesque and Chantal Guillemette
The prognostic significance of common deletions in uridine diphospho-glucuronosyltransferase 2B (UGT2B) genes encoding sex steroid metabolic enzymes has been recently recognized in localized prostate cancer (PCa) after radical prostatectomy (RP). However, the role of germline variations at the UGT1 locus, encoding half of all human UGTs and primarily involved in estrogen metabolism, remains unexplored. We investigated whether variants of UGT1 are potential prognostic markers. We studied 526 Caucasian men who underwent RP for clinically localized PCa. Genotypes of patients for 34 haplotype-tagged single-nucleotide polymorphisms (htSNPs) and 11 additional SNPs across the UGT1 locus previously reported to mark common variants including functional polymorphisms were determined. The risk of biochemical recurrence (BCR) was estimated using adjusted Cox proportional hazards regression and Kaplan–Meier analysis. We further investigated whether variants are associated with plasma hormone levels by mass spectrometry. In multivariable models, seven htSNPs were found to be significantly associated with BCR. A greater risk was revealed for four UGT1 intronic variants with hazard ratios (HRs) of 1.59–1.88 (P<0.002) for htSNPs in UGT1A10, UGT 1A9, and UGT1A6. Conversely, decreased BCR was associated with three htSNPs in introns of UGT1A10 and UGT1A9 (HR=0.56–058; P≤0.01). An unfavorable UGT1 haplotype comprising all risk alleles, with a frequency of 14%, had a HR of 1.68 (95% CI=1.13–2.50; P=0.011). Significant alteration in circulating androsterone levels was associated with this haplotype, consistent with changes in hormonal exposure. This study provides the first evidence, to our knowledge, that germline polymorphisms of UGT1 are potential predictors of recurrence of PCa after prostatectomy.
Arvind Dasari, Alexandria Phan, Sanjay Gupta, Asif Rashid, Sai-Ching Jim Yeung, Kenneth Hess, Helen Chen, Emily Tarco, Huiqin Chen, Caimiao Wei, Kim Anh-Do, Daniel Halperin, Funda Meric-Bernstam and James Yao
Preclinical data suggest multiple roles for the IGF1 receptor (IGF1R) in neuroendocrine tumors (NETs), including mediating resistance to mammalian target of rapamycin (mTOR) inhibitors. Everolimus, an oral mTOR inhibitor, and octreotide long-acting repeatable (LAR) are approved for subgroups of well-differentiated NET. The primary objective of the present study was to establish the safety and recommended phase II dose (RP2D) of cixutumumab, a monoclonal antibody (MAB) against IGF1R, with everolimus and octreotide LAR. Patients with well-differentiated NET were treated with 10 mg everolimus p.o. daily, 20 mg octreotide LAR i.m. every 21 days, and escalating doses of cixutumumab. An expansion cohort was enrolled at RP2D. Correlative studies included the evaluation of mTOR pathway inhibition in paired tumor biopsies and the effects of this combination on metabolism via indirect calorimetry. Nineteen patients with progressive disease were enrolled, including nine to the expansion portion. Two patients had dose-limiting toxicities of grade 3 mucositis at 15 mg/kg cixutumumab. Long-term tolerance at RP2D was problematic, and the most common ≥grade 3 adverse event was fatigue. One patient with metastatic insulinoma had a confirmed partial response, whereas 17 had stable disease. The median progression-free survival was 43.6 weeks, and the median overall survival was 25.5 months. The RP2D of this combination per the predefined study protocol of 10 mg/kg cixutumumab i.v., 20 mg octreotide LAR i.m. every 21 days plus 10 mg everolimus p.o. daily is associated with non-dose-limiting toxicities that limit long-term tolerance. Although a signal of activity was noted in the present study, this will need to be reconciled with limited tolerance of the combination and data from larger studies of anti-IGF1R MABs in NET that have been disappointing.
D M Peehl
This review focuses on primary cultures of human prostatic epithelial cells and their applications as models of normal and malignant biological behavior. Current abilities to culture cells from normal tissues, from premalignant dysplastic lesions (prostatic intraepithelial neoplasia), from primary adenocarcinomas, and from metastases are described. Evidence for representation of the interrelated cells of the normal prostatic epithelium — stem cells, basal epithelial cells, secretory epithelial cells, transit amplifying cells and neuroendocrine cells — in primary cultures is presented. Comparisons between normal and cancer-derived primary cultures are made regarding biological activities relevant to carcinogenesis, such as proliferation, apoptosis, differentiation, senescence, adhesion, migration, invasion, steroid hormone metabolism, other metabolic pathways and angiogenesis. Analyses of tumor suppressor activity, differential gene expression and cytogenetics in primary cultures have revealed changes relevant to prostate cancer progression. Preclinical studies with primary cultures have provided information useful for designing new strategies for chemoprevention, chemotherapy, cytotoxin therapy, radiation therapy, gene therapy and imaging. While the behavior of normal primary cultures is often used as a basis for comparison with established, immortal prostate cancer cell lines, the most informative studies are performed with donor-matched pairs of normal and malignant primary cultures, grown under identical conditions. Challenges that remain to be addressed if the full potential of primary cultures as a model system is to be realized include isolation, culture and characterization of stem cells, improved methodology to induce or maintain a fully differentiated, androgen-responsive phenotype, and identification of cell surface antigens or other markers with which to purify pure populations of live cancer or premalignant cells apart from non-malignant epithelial cells prior to culture.
S Leskelä, E Honrado, C Montero-Conde, I Landa, A Cascón, R Letón, P Talavera, J M Cózar, A Concha, M Robledo and C Rodríguez-Antona
Testosterone is essential for the growth and function of the luminal prostate cells, but it is also critical for the development of prostate cancer, which in the majority of the cases derives from luminal cells. Cytochrome P450 3A (CYP3A) enzymes hydroxylate testosterone and dehydroepiandrosterone to less active metabolites, which might be the basis for the association between CYP3A polymorphisms and prostate cancer. However, it is unknown whether the CYP3A enzymes are expressed at relevant levels in the prostate and which polymorphisms could affect this tissue-specific CYP3A activity. Thus, we measured CYP3A4, CYP3A5, CYP3A7, and CYP3A43 mRNA in 14 benign prostatic hyperplasias and ten matched non-tumoral/tumoral prostate samples. We found that CYP3A5 mRNA in non-tumoral prostate tissue was 10% of the average amount of liver samples, whereas the expression of the other CYP3A genes was much lower. Similarly to liver, CYP3A5*3 polymorphism decreased CYP3A5 mRNA content 13-fold. CYP3A5 protein was detected in non-tumoral prostate microsomes by western blot, and immunohistochemistry (IHC) localized CYP3A5 exclusively in the basolateral prostate cells. In contrast to the normal tissue, IHC and RT-PCR showed that tumoral tissue lacked CYP3A5 expression. In conclusion, prostate basolateral cells express high levels of CYP3A5 which dramatically decrease in tumoral tissue. This finding supports an endogenous function of CYP3A5 related to the metabolism of intra-prostatic androgens and cell growth, and that polymorphisms affecting CYP3A5 activity may result in altered prostate cancer risk and aggressiveness.