Parathyroid hormone‐related protein confers chemoresistance by blocking apoptosis signaling via death receptors and mitochondria

Parathyroid hormone (PTH) has a central role in the regulation of serum calcium and phosphate, whereas parathyroid hormone‐related peptide (PTHrP) has important developmental roles. In addition, PTHrP has been discovered as a causative agent of hypercalcemia of malignancy. PTHrP is also expressed in many tumors, and expression often correlates with unfavorable prognosis. We have investigated the effects of PTHrP on apoptosis signaling pathways initiated by DNA damaging chemotherapeutic drugs. Stimulation experiments of the CD95‐, the TNF‐R‐, and the TRAIL‐R‐death receptor systems in Saos human osteosarcoma cells revealed that PTHrP can block signaling via each of these death receptors. Furthermore, our findings demonstrate a link between PTHrP and the mitochondrial apoptosis pathway. PTHrP down‐regulates expression of pro‐apoptotic Bcl‐2 family members like Bax and PUMA and up‐regulates expression of antiapoptotic molecules like Bcl‐2 and Bcl‐xl. It is of clinical relevance that PTHrP and anticancer drugs show opposing interactions on death receptor‐triggered as well as on mitochondrial apoptosis pathways. In addition, PTHrP induces chemoresistance by interference with p53 family‐dependent apoptosis signaling pathways and p53‐mediated transactivation of apoptosis target genes. Inhibition of CD95‐ and Bax gene transactivation is a mechanism by which PTHrP reduced the apoptosis response and treatment sensitivity of tumor cells. Our data indicate that PTHrP inhibits major apoptosis signaling pathways by blocking signaling via p53, death receptors and mitochondria and, consequently, confers chemoresistance of cancer cells. Thus, beyond its importance in development and differentiation, we describe an important role for PTHrP in tumorigenesis. © 2009 UICC     

Capecitabine improves cancer cachexia and normalizes IL-6 and PTHrP levels in mouse cancer cachexia models

Purpose

To clarify the potential of parathyroid hormone-related protein (PTHrP) and interleukin-6 (IL-6) as cachectic factors in a colon 26 model and the effects of capecitabine on cancer cachexia as determined by plasma levels of IL-6 and PTHrP and body weight loss.

Methods

From two colon 26 sublines-cancer cachectic clone20 and non-cachectic clone5 plasma levels of PTHrP protein and mRNA expression levels in tumor tissues were compared. An IL-6 neutralizing antibody, a PTHrP neutralizing antibody, and capecitabine were administered into mice bearing clone20 and their anticachectic effects evaluated.

Results

The plasma level of PTHrP protein in mice bearing clone20 was higher than that in mice bearing clone5. The expression level of PTHrP mRNA was 49-fold higher in tumor tissues of clone20 than of clone5, according to GeneChip^® analysis. PTHrP antibody as well as IL-6 antibody suppressed wasting of the body and gastrocnemius and adipose tissue weights. PTHrP antibody suppressed the induction of hypercalcemia but not hypoglycemia or elevation of IL-6, whereas IL-6 antibody suppressed the induction of hypoglycemia but not hypercalcemia or elevation of PTHrP. Capecitabine, a fluorinated pyrimidine anticancer agent, improved body wasting of mice bearing clone20 at a low dose with no reduction of tumor volume. Furthermore, capecitabine lowered the levels of PTHrP and IL-6 in plasma and suppressed hypoglycemia and hypercalcemia in this model. Capecitabine also showed anticachectic effects on cachexia in a cancer model induced by human cervical cancer cell line Y (also known as Yumoto).

Conclusions

PTHrP and IL-6 were found to be factors in the development of cachexia in a colon 26 cancer model, and capecitabine improved cancer cachexia by suppressing the plasma levels of IL-6 and PTHrP in colon 26 and Y cachectic models.     

Identification of HLA‐A2‐ and A24‐restricted T‐cell epitopes derived from SOX6 expressed in glioma stem cells for immunotherapy

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma‐bearing mice. In this study, we attempted to identify SOX6‐derived peptides as specific targets for effective and safe T‐cell‐mediated immunotherapy targeting SOX6‐positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)‐A*2402 (A24)‐restricted peptides, RFENLGPQL (SOX6₅₀₄) and PYYEEQARL (SOX6₆₂₈) or the HLA‐A*0201 (A2)‐restricted peptide, ALFGDQDTV (SOX6₄₄₇) was capable of inducing SOX6 peptide‐specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I‐restricted and an antigen‐dependent manner. Furthermore, peptide vaccines of SOX6₆₂₈, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H‐2^d, induced CTLs specific for SOX6₆₂₈ in H‐2^d mice. Normal autologous cells from mice, in which SOX6‐specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA‐A24 or ‐A2 positive glioma patients and should be considered as a promising strategy for safe and effective T‐cell‐based immunotherapy of patients with gliomas.     

A MAGE‐1‐encoded HLA‐A24‐binding synthetic peptide induces specific anti‐tumor cytotoxic T lymphocytes

Although several MAGE‐1 peptides have already been identified, the MAGE‐1‐encoded peptide presented by HLA‐A24, which is the most common allele in Japanese population and is also frequently present in Caucasians, might have a wide applicability for immunotherapy using these peptides. To identify this potential peptide, we examined the induction of specific cytotoxic T lymphocytes (CTL) from the peripheral‐blood mononuclear cells (PBMC) in HLA‐A24 healthy donors by in vitro stimulation with MAGE‐1‐encoded synthetic peptides with a binding affinity for HLA‐A24, by a simplified method. Of the 5 peptides tested, the highest HLA binder (NYKHCFPEI) was able to elicit CTL from unseparated PBMC by stimulation with freshly isolated, peptide‐pulsed PMBC as antigen‐presenting cells (APC) and by also using interleukin 7 and keyhole‐limpet hemocyanin for a primary culture. The induced CTL could thus lyse HLA‐A24 tumor cells expressing MAGE‐1, as well as the peptide‐pulsed target cells, in an HLA‐class‐I‐restricted manner. By using the MAGE‐1/HLA‐A24 peptide, NYKHCFPEI, we found it possible to immunize many more patients, especially Japanese patients, by means of such peptide‐based immunotherapeutic approaches to MAGE‐1‐positive malignant tumors. Int. J. Cancer 80:169–172, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of CDCA1‐derived long peptides bearing both CD4+ and CD8+ T‐cell epitopes: CDCA1‐specific CD4+ T‐cell immunity in cancer patients

We recently identified a novel cancer‐testis antigen, cell division cycle associated 1 (CDCA1) using genome‐wide cDNA microarray analysis, and CDCA1‐derived cytotoxic T lymphocyte (CTL)‐epitopes. In this study, we attempted to identify CDCA1‐derived long peptides (LPs) that induce both CD4⁺ helper T (Th) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II‐binding peptides with CDCA1‐derived CTL‐epitope sequences presented by HLA‐A2 (A*02:01) or HLA‐A24 (A*24:02) to select candidate CDCA1‐LPs encompassing both Th cell epitopes and CTL‐epitopes. We studied the immunogenicity of CDCA1‐LPs and the cross‐priming potential of LPs bearing CTL‐epitopes in both human in vitro and HLA‐class I transgenic mice in vivo. Then we analyzed the Th cell response to CDCA1 in head‐and‐neck cancer (HNC) patients before and after vaccination with a CDCA1‐derived CTL‐epitope peptide using IFN‐γ enzyme‐linked immunospot assays. We identified two CDCA1‐LPs, CDCA1_39–64‐LP and CDCA1_55–78‐LP, which encompass naturally processed epitopes recognized by Th cells and CTLs. CDCA1‐specific CTLs were induced through cross‐presentation of CDCA1‐LPs in vitro and in vivo. In addition, CDCA1‐specific Th cells enhanced induction of CDCA1‐specific CTLs. Furthermore, significant frequencies of CDCA1‐specific Th cell responses were detected after short‐term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with CDCA1‐LPs in HNC patients (CDCA1_39–64‐LP, 74%; CDCA1_55–78‐LP, 68%), but not in healthy donors. These are the first results demonstrating the presence of CDCA1‐specific Th cell responses in HNC patients and underline the possible utility of CDCA1‐LPs for propagation of both CDCA1‐specific Th cells and CTLs.     

Identification of a rhabdomyosarcoma targeting peptide by phage display with sequence similarities to the tumour lymphatic‐homing peptide LyP‐1

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. To improve existing therapies and broaden the spectrum of cytotoxic agents that can be used in RMS treatment, we performed a phage‐display‐based screening for peptides that bind specifically to RMS cells. Two peptides binding to RMS and to other tumour cell lines, but not to normal skeletal muscle cells and fibroblasts, were isolated from phage‐displayed random peptide libraries. One peptide, named RMS‐I (CQQSNRGDRKRC) contained the integrin‐binding motif RGD and its binding was blocked by an antibody against α_vβ₃integrin, which is expressed on the RMS cell line RD. The isolation of RMS‐I confirmed the validity of our screening procedure. The second peptide, named RMS‐II (CMGNKRSAKRPC), shows sequence similarity to a previously identified peptide with tumour lymphatic specificity, LyP‐1. However, RMS‐II binds in vivo to RMS xenografts better than LyP‐1 and homes to the tumour blood and not to lymphatic vessels. Therefore, RMS‐II represents a promising peptide for the development of RMS‐specific targeting approaches. © 2008 Wiley‐Liss, Inc.     

Organotropic Chemopreventive Effects of n-3 Unsaturated Fatty Acids in a Rat Multi-organ Carcinogenesis Model

Organotropic chemopreventive effects of n‐3 unsaturated fatty acids were studied using a multi‐organ carcinogenesis model in male rats. Rats were treated with diethylnitrosamine (DEN), N‐methyl‐N‐nitrosourea (MNU), N‐butyl‐N‐4‐hydroxybutylnitrosamine (BBN), 1,2‐dimethylhydrazine (DMH) and dihydroxy‐di‐n‐propylnitrosamine (DHPN) during the first 7 weeks, and then given unsaturated fatty acid (UFAs), docosahexaenoic acid (n‐3, C_22:6) (DHA), eicosapentaenoic acid (n‐3, C_20:5) (EPA), linoleic acid (n‐6, C_18:2) (LA) or oleic acid (n‐9, C_18:1) (OA) at a dose of 1.0 ml/rat, 3 tunes a week by gavage for the consecutive 30 weeks. All rats were fed a low LA basal diet throughout the experiment and a calorie‐restricted basal diet during the period of UFAs feeding administration. DHA significantly reduced tumor size and numbers in the large intestine as compared to OA treatment. Furthermore, DHA showed a tendency to inhibit carcinogenesis in the small intestine and lung. EPA also showed a tendency to inhibit intestinal carcinogenesis. On the other hand, LA showed a tendency to inhibit lung carcinogenesis, but to promote large intestinal carcinogenesis. However these UFAs did not influence preneoplastic and neoplastic lesion development in the liver, kidney, and urinary bladder. Levels of the administered fatty acids were clearly increased in the serum and organs. In contrast, arachidonic acid (AA) levels in the large and small intestines and liver were markedly decreased by treatment with DHA and EPA. Decreased levels of AA in the large intestine correlated well with tumor incidence, although the number of glutathione S‐transferase‐positive (GST‐P⁺) foci showed an inverse correlation with AA levels. The data thus provide evidence that an organotropism exists with regard to the influence of UFAs on carcinogenesis, which correlates with reduction of tissue AA levels in the target organs.     

Cancer anorexia-cachexia syndrome: are neuropeptides the key?

Progressive wasting is common in many types of cancer and is one of the most important factors leading to early death in cancer patients. Weight loss is a potent stimulus to food intake in normal humans and animals. The persistence of anorexia in cancer patients, therefore, implies a failure of this adaptive feeding response, although the weight loss in the patients differs from that found in simple starvation. Tremendous progress has been made in the last 5 years with regard to the regulation of feeding and body weight. It has been demonstrated that leptin, a hormone secreted by adipose tissue, is an integral component of the homeostatic loop of body weight regulation. Leptin acts to control food intake and energy expenditure via neuropeptidergic effector molecules within the hypothalamus. Complex interactions among the nervous, endocrine, and immune systems affect the loop and induce behavioral and metabolic responses. A number of cytokines, including tumor necrosis factor-alpha, interleukins 1 and 6, IFN-gamma, leukemia inhibitory factor, and ciliary neurotrophic factor have been proposed as mediators of the cachectic process. Cytokines may play a pivotal role in long-term inhibition of feeding by mimicking the hypothalamic effect of excessive negative feedback signaling from leptin. This could be done by persistent stimulation of anorexigenic neuropeptides such as corticotropin-releasing factor, as well as by inhibition of the neuropeptide Y orexigenic network that consists of opioid peptides and galanin, in addition to the newly identified melanin-concentrating hormone, orexin, and agouti-related peptide. Information is being gathered, although it is still insufficient, on such abnormalities in the hypothalamic neuropeptide circuitry in tumor-bearing animals that coincide with the development of anorexia and cachexia. Characterization of the feeding-associated gene products have revealed new biochemical pathways and molecular targets for pharmacological intervention that will likely lead to new treatments. Although therapeutic intervention using neuropeptide agonists/antagonists is now directed at obesity treatment, it may also have an effect on treating cancer anorexia-cachexia, especially when combined with other agents that have effects on muscle and protein breakdown.     

Delta‐6‐desaturase activity and arachidonic acid synthesis are increased in human breast cancer tissue

Omega‐6 (n‐6) arachidonic acid (AA) and its pro‐inflammatory metabolites, including prostaglandin E2 (PGE₂), are known to promote tumorigenesis. Delta‐6 desaturase (D6D) is the rate‐limiting enzyme for converting n‐6 linoleic acid (LA) to AA. Our objective was to determine if AA synthesis, specifically D6D activity, and PGE₂ levels are increased in cancerous breast tissue, and whether these variables differ between estrogen receptor positive (ER+) and negative (ER?) breast cancers. Gas chromatography was performed on surgical breast tissue samples collected from 69 women with breast cancer. Fifty‐four had ER+ breast cancer, and 15 had ER? breast cancer. Liquid chromatography‐mass spectrometry was used to determine PGE₂ levels. Lipid analysis revealed higher levels of LA metabolites (C18:3 n‐6, C20:3 n‐6, and AA) in cancerous tissue than in adjacent noncancerous tissue (P < 0.01). The ratio of LA metabolites to LA, a measure of D6D activity, was increased in cancerous tissue, suggesting greater conversion of LA to AA (P < 0.001), and was higher in ER? than in ER+ patients, indicating genotype‐related trends. Similarly, PGE₂ levels were increased in cancerous tissue, particularly in ER? patients. The results showed that the endogenous AA synthetic pathway, D6D activity, and PGE₂ levels are increased in breast tumors, particularly those of the ER? genotype. These findings suggest that the AA synthetic pathway and the D6D enzyme in particular may be involved in the pathogenesis of breast cancer. The development of drugs and nutritional interventions to alter this pathway may provide new strategies for breast cancer prevention and treatment.     

Hypercalcemia Associated with a Malignant Brenner Tumor Arising from a Mature Cystic Teratoma

A 60-year-old woman presented with abdominal pain and weight loss and was found to have serum calcium of 15.0 mg/dl. Serum parathyroid hormone-related peptide (PTHrP) returned elevated. Imaging suggested bilateral mature cystic teratomas. Her hypercalcemia was treated initially with intravenous saline, as well as intramuscular and subcutaneous calcitonin. She underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy, and final pathology revealed malignant Brenner tumor in association with a mature cystic teratoma. Her postoperative PTHrP returned less than assay, and her total and ionized calcium fell below normal, requiring supplemental calcium and vitamin D. At follow-up one month after discharge, her calcium had normalized. We present the first reported case of hypercalcemia occurring in association with a malignant Brenner tumor. Malignancy-associated hypercalcemia occurs via four principal mechanisms: (1) tumor production of PTHrP; (2) osteolytic bone involvement by primary tumor or metastasis; (3) ectopic activation of vitamin D to 1,25-(OH)₂ vitamin D, and (4) ectopic production of parathyroid hormone. PTHrP-mediated hypercalcemia is the most common mechanism and was responsible in this case. In patients with paraneoplastic hypercalcemia who undergo surgical treatment, close monitoring and management of serum calcium is necessary both pre- and postoperatively.Key Words: Hypercalcemia, Parathyroid hormone-related peptide, Mature cystic teratoma, Malignant Brenner tumor     

Phase‐1 study of abiraterone acetate in chemotherapy‐naïve Japanese patients with castration‐resistant prostate cancer

Persistent androgen synthesis under castration status in adrenal gland, testes and tumor cells is thought to be one of the major causes of development and progression of castration‐resistant prostate cancer (CRPC). Abiraterone acetate (AA), the prodrug of abiraterone, which is an inhibitor of androgen synthesis enzymes, was evaluated for pharmacokinetics, pharmacodynamics, preliminary efficacy and safety in Japanese patients with CRPC in a phase‐1, open‐label and dose‐escalation study. Chemotherapy‐naïve Japanese CRPC patients (N = 27) received one of four AA daily doses (250 mg [n = 9], 500 mg [n = 6], 1000 [1 h premeal] mg [n = 6] and 1000 [2 h postmeal] mg [n = 6]) continuously through 28‐day treatment cycles. In the first cycle, AA monotherapy was given on days 1–7 for pharmacokinetics, and AA plus prednisone (5 mg twice daily) from days 8 to 28. Of 27 patients, 9 continued treatment with AA until the data cut‐off date (18 July 2013). Over the evaluated dose range, plasma abiraterone concentrations increased with dose, with median t_max 2–3 h. At each dose level, mean serum corticosterone concentrations increased, while testosterone and dehydroepiandrosterone sulfate concentrations rapidly decreased following a single AA dose and were further reduced to near the quantification limit on day 8 regardless of the dose. At least 3 patients from each dose‐group experienced ≥50% prostate‐specific antigen reduction, suggesting clinical benefit from AA in Japanese CRPC patients. AA was generally well‐tolerated, and, therefore, the recommended AA dosage regimen in Japanese CRPC patients is 1000 mg oral dose under modified fasting conditions (at least 1 h premeal or 2 h postmeal). This study is registered at ClinicalTrials.gov : NCT01186484.     

甲状旁腺素相关肽与肿瘤

甲状旁腺素相关肽(PTHrP)参与调节钙磷代谢和多器官的生长发育,在恶性肿瘤发生骨转移和高钙血症过程中发挥重要作用.越来越多的研究证实PTHrP可以由多种类型的肿瘤细胞分泌,参与调节肿瘤细胞的增殖、侵袭,并与患者预后密切相关,为肿瘤治疗提供了新的靶点.     

Discovery of inner centromere protein‐derived small peptides for cancer imaging and treatment targeting survivin

Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer‐specific treatment. In this study, we designed and synthesized 7‐19 residues of inner centromere protein (INCENP)‐derived small peptides (INC peptides) as novel survivin‐targeting agents. The INC peptides showed binding affinity for the human survivin protein (K_d = 91.4‐255 nmol L^?1); INC_16‐22, which contains residues 16‐22 of INCENP, showed the highest affinity (91.4 nmol L^?1). Confocal fluorescence imaging showed consistent colocalization of FITC‐INC_16‐22 and survivin in cell lines. Nona‐arginine‐linked INC_16‐22 (r9‐INC_16‐22) rendered INC_16‐22 cells penetrable and strongly inhibited cell growth of MIA PaCa‐2 cells (52% inhibition at 1.0 µmol L^?1) and MDA‐MB‐231 cells (60% inhibition at 10 µmol L^?1) as determined by MTT assays. The exposure of MIA PaCa‐2 cells to 40 µmol L^?1 r9‐INC_16‐22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase‐3 was significantly increased in cells treated with r9‐INC_16‐22, even at 10 µmol L^?1, compared to untreated cells. Flow cytometry revealed that r9‐INC_16‐22 strongly induced apoptosis in MIA PaCa‐2 cells. These results indicate that the cytotoxic effects of r9‐INC_16‐22 could be mediated mainly through the disruption of survivin‐dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.     

Evaluation of a new oil adjuvant for use in peptide‐based cancer vaccination

Vaccine therapies are increasingly being used for the treatment of various diseases, and the antigen molecules themselves are being expanded from whole microorganisms to fine molecules such as peptides. Accordingly, there is a need for new adjuvants to support these new applications. In this paper, we used pharmaceutical grade mineral oil and sorbitan monooleate to develop a new oil adjuvant formula, NH₂, and investigated its effects on peptide vaccination at both the pre‐clinical and clinical levels. The adjuvant effect of NH₂ on peptide‐induced cellular immunity in mice was superior to that of Montanide ISA51VG, a commercially available incomplete Freund’s adjuvant for clinical use, although no significant difference was observed between the two adjuvants on peptide‐induced humoral immunity. The adjuvant effects of NH₂ were also confirmed in a Phase‐I clinical trial of peptide vaccines for patients with advanced cancers. These results suggest that NH₂ is a suitable adjuvant for peptide vaccination, particularly for cancer vaccines (Phase‐I clinical trial of pan‐HLA type personalized peptide vaccine for advanced cancer patients, UMIN clinical trial registry number: UMIN 000000619). (Cancer Sci 2010)     

Dietary‐feeding of grape seed extract prevents azoxymethane‐induced colonic aberrant crypt foci formation in fischer 344 rats

Chemoprevention by dietary agents/supplements has emerged as a novel approach to control various malignancies, including colorectal cancer (CRC). This study assessed dietary grape seed extract (GSE) effectiveness in preventing azoxymethane (AOM)‐induced aberrant crypt foci (ACF) formation and associated mechanisms in Fischer 344 rats. Six‐week‐old rats were injected with AOM, and fed control diet or the one supplemented with 0.25% or 0.5% (w/w) GSE in pre‐ and post‐AOM or only post‐AOM experimental protocols. At 16 wk of age, rats were sacrificed and colons were evaluated for ACF formation followed by cell proliferation, apoptosis, and molecular analyses by immunohistochemistry. GSE‐feeding caused strong chemopreventive efficacy against AOM‐induced ACF formation in terms of up to 60% (P < 0.001) reduction in number of ACF and 66% (P < 0.001) reduction in crypt multiplicity. Mechanistic studies showed that GSE‐feeding inhibited AOM‐induced cell proliferation but enhanced apoptosis in colon including ACF, together with a strong decrease in cyclin D1, COX‐2, iNOS, and survivin levels. Additional studies showed that GSE‐feeding also decreased AOM‐caused increase in β‐catenin and NF‐κB levels in colon tissues. Compared to control animals, GSE alone treatment did not show any considerable change in these biological and molecular events in colon, and was nontoxic. Together, these findings show the chemopreventive efficacy of GSE against the early steps of colon carcinogenesis in rats via likely targeting of β‐catenin and NF‐κB signaling, and suggest its potential usefulness for the prevention of human CRC. © 2010 Wiley‐Liss, Inc.     

Oncogenic microRNA‐27a is a target for anticancer agent methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate in colon cancer cells

Methyl 2‐cyano‐3,11‐dioxo‐18β‐olean‐1,12‐dien‐30‐oate (CDODA‐Me) is a synthetic derivative of glycyrrhetinic acid, a triterpenoid phytochemical found in licorice extracts. CDODA‐Me inhibited growth of RKO and SW480 colon cancer cells and this was accompanied by decreased expression of Sp1, Sp3 and Sp4 protein and mRNA and several Sp‐dependent genes including survivin, vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR1 or Flt‐1). CDODA‐Me also induced apoptosis, arrested RKO and SW480 cells at G₂/M, and inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. CDODA‐Me decreased expression of microRNA‐27a (miR‐27a), and this was accompanied by increased expression of 2 miR‐27a‐regulated mRNAs, namely ZBTB10 (an Sp repressor) and Myt‐1 which catalyzes phosphorylation of cdc2 to inhibit progression of cells through G₂/M. Both CDODA‐Me and antisense miR‐27a induced comparable responses in RKO and SW480 cells, suggesting that the potent anticarcinogenic activity of CDODA‐Me is due to repression of oncogenic miR‐27a. © 2009 UICC     

Serum insulin‐like growth factor (IGF)‐I and IGF binding protein‐3 in relation to terminal duct lobular unit involution of the normal breast in Caucasian and African American women: The Susan G. Komen Tissue Bank

Lesser degrees of terminal duct lobular unit (TDLU) involution, as reflected by higher numbers of TDLUs and acini/TDLU, are associated with elevated breast cancer risk. In rodent models, the insulin‐like growth factor (IGF) system regulates involution of the mammary gland. We examined associations of circulating IGF measures with TDLU involution in normal breast tissues among women without precancerous lesions. Among 715 Caucasian and 283 African American (AA) women who donated normal breast tissue samples to the Komen Tissue Bank between 2009 and 2012 (75% premenopausal), serum concentrations of IGF‐I and binding protein (IGFBP)‐3 were quantified using enzyme‐linked immunosorbent assay. Hematoxilyn and eosin‐stained tissue sections were assessed for numbers of TDLUs (“TDLU count”). Zero‐inflated Poisson regression models with a robust variance estimator were used to estimate relative risks (RRs) for association of IGF measures (tertiles) with TDLU count by race and menopausal status, adjusting for potential confounders. AA (vs. Caucasian) women had higher age‐adjusted mean levels of serum IGF‐I (137 vs. 131 ng/mL, p = 0.07) and lower levels of IGFBP‐3 (4165 vs. 4684 ng/mL, p < 0.0001). Postmenopausal IGFBP‐3 was inversely associated with TDLU count among AA (RR_T3vs.T1 = 0.49, 95% CI = 0.28–0.84, p‐trend = 0.04) and Caucasian (RR_T3vs.T1=0.64, 95% CI = 0.42–0.98, p‐trend = 0.04) women. In premenopausal women, higher IGF‐I:IGFBP‐3 ratios were associated with higher TDLU count in Caucasian (RR_T3vs.T1=1.33, 95% CI = 1.02–1.75, p‐trend = 0.04), but not in AA (RR_T3vs.T1=0.65, 95% CI = 0.42–1.00, p‐trend = 0.05), women. Our data suggest a role of the IGF system, particularly IGFBP‐3, in TDLU involution of the normal breast, a breast cancer risk factor, among Caucasian and AA women.     

Immunological evaluation of peptide vaccination for cancer patients with the HLA‐A26 allele

To develop a peptide vaccine for cancer patients with the HLA‐A26 allele, which is a minor population worldwide, we investigated the immunological responses of HLA‐A26⁺/A26⁺ cancer patients to four different CTL epitope peptides under personalized peptide vaccine regimens. In personalized peptide vaccine regimens, two to four peptides showing positive peptide‐specific IgG responses in pre‐vaccination plasma were selected from the four peptide candidates applicable for HLA‐A26⁺/A26⁺ cancer patients and administered s.c. Peptide‐specific CTL and IgG responses along with cytokine levels were measured before and after vaccination. Cell surface markers in PBMCs and plasma cytokine levels were also measured. In this study, 21 advanced cancer patients, including seven lung, three breast, two pancreas, and two colon cancer patients, were enrolled. Their HLA‐A26 genotypes were HLA‐A26:01 (n = 24), HLA‐A26:03 (n = 10), and HLA‐A26:02 (n = 8). One, 14, and 6 patients received two, three, and four peptides, respectively. Grade 1 or 2 skin reactions at the injection sites were observed in the majority of patients, but no severe adverse events related to the vaccination were observed. Peptide‐specific CTL responses were augmented in 39% or 22% of patients after one or two cycles of vaccination, respectively. Notably, peptide‐specific IgG were augmented in 63% or 100% of patients after one or two cycles of vaccination, respectively. Personalized peptide vaccines with these four CTL epitope peptides could be feasible for HLA‐A26⁺ advanced cancer patients because of their safety and higher rates of immunological responses.     

The calcium‐sensing receptor and parathyroid hormone‐related protein are expressed in differentiated,favorable neuroblastic tumors

BACKGROUND:

Differentiated histopathology is a favorable prognostic factor in neuroblastic tumors, and molecular pathways underlying neuroblastoma differentiation can be modulated pharmacologically. The calcium‐sensing receptor (CaR) and parathyroid hormone‐related protein (PTHrP) regulate differentiation processes in some cellular contexts. CaR is up‐regulated when neural stem cells are specified to the oligodendrocyte lineage and regulates PTHrP production in astrocytes. The objective of the current study was to assess whether CaR and PTHrP participate in neuroblastoma differentiation pathways.

METHODS:

CaR and PTHrP messenger RNA (mRNA) and protein expression were analyzed in neuroblastic tumors, and correlation with prognostic factors was assessed. CaR and PTHrP expression levels were analyzed in neuroblastoma cell lines treated with all‐trans‐retinoic acid or 5‐bromo‐2′‐deoxyuridine (BrdU).

RESULTS:

CaR expression was correlated with favorable histology, age at diagnosis <1 year, low clinical stage, and low clinical risk. CaR was absent in undifferentiated neuroblasts and was expressed in differentiating neuroblasts. CaR and PTHrP were highly expressed in ganglion and in Schwann‐like cells. PTHrP mRNA levels were higher in ganglioneuroblastomas and ganglioneuromas than in neuroblastomas (P < .0001). Both genes were up‐regulated in neuroblastomas with treatment‐induced maturation features. CaR, but not PTHrP, was up‐regulated at early phases of in vitro neuronal differentiation induction. Substrate‐adherent, non‐neuronal cell lines displayed the highest PTHrP levels among the neuroblastoma cell lines examined. The up‐regulation of PTHrP and of 2 glial differentiation markers was observed in 2 cell lines that were treated with BrdU, whereas CaR was induced in only 1 cell line.

CONCLUSIONS:

CaR and PTHrP were expressed in differentiated, favorable neuroblastic tumors, and both genes were up‐regulated by inducing differentiation. Cancer 2009. © 2009 American Cancer Society.