Non‐invasive estimation of 10B‐4‐borono‐L‐phenylalanine‐derived boron concentration in tumors by PET using 4‐borono‐2‐18F‐fluoro‐phenylalanine

In boron neutron capture therapy (BNCT), ¹⁰B‐4‐borono‐L‐phenylalanine (BPA) is commonly used as a ¹⁰B carrier. PET using 4‐borono‐2‐¹⁸F‐fluoro‐phenylalanine (¹⁸F‐FBPA PET) has been performed to estimate boron concentration and predict the therapeutic effects of BNCT; however, the association between tumor uptake of ¹⁸F‐FBPA and boron concentration in tumors remains unclear. The present study investigated the transport mechanism of ¹⁸F‐FBPA and BPA, and evaluated the utility of ¹⁸F‐FBPA PET in predicting boron concentration in tumors. The transporter assay revealed that 2‐aminobicyclo‐(2.2.1)‐heptane‐2‐carboxylic acid, an inhibitor of the L‐type amino acid transporter, significantly inhibited ¹⁸F‐FBPA and ¹⁴C‐4‐borono‐L‐phenylalanine (¹⁴C‐BPA) uptake in FaDu and LN‐229 human cancer cells. ¹⁸F‐FBPA uptake strongly correlated with ¹⁴C‐BPA uptake in 7 human tumor cell lines (r = .93; P < .01). PET experiments demonstrated that tumor uptake of ¹⁸F‐FBPA was independent of the administration method, and uptake of ¹⁸F‐FBPA by bolus injection correlated well with BPA uptake by continuous intravenous infusion. The results of this study revealed that evaluating tumor uptake of ¹⁸F‐FBPA by PET was useful for estimating ¹⁰B concentration in tumors.     

Attenuation by genistein of sodium‐chloride‐enhanced gastric carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine in Wistar rats

The effects of prolonged administration of genistein, a tyrosine‐kinase inhibitor, on sodium‐chloride‐enhanced induction of gastric carcinogenesis induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, and the labeling and apoptotic indices and vessel counts in the gastric mucosa and gastric cancers, were investigated in Wistar rats. After 25 weeks of the carcinogen treatment, rats were fed chow pellets containing 10% sodium chloride and were given s.c. injections of genistein at dosages of 15 mg/kg or 30 mg/kg body weight every other day. In week 52, the incidence of gastric cancers was significantly greater in rats fed sodium chloride than in untreated control rats. Prolonged administration of genistein at a dosage of 30 mg/kg, but not 15 mg/kg, body weight significantly reduced the incidence of gastric cancers, which was increased by oral treatment with sodium chloride. Genistein at the higher dose significantly decreased the labeling index and vessel counts of the antral mucosa and the gastric cancers (which were increased by treatment with sodium chloride) and significantly increased the apoptotic index of the antral mucosa and the cancers (which was lowered by the treatment with sodium chloride). These findings suggest that genistein attenuates gastric carcinogenesis promoted by sodium chloride, by inducing increased apoptosis and lower cell proliferation and angiogenesis of antral mucosa and gastric cancers. Int. J. Cancer 80:396–399, 1999. © 1999 Wiley‐Liss, Inc.     

Melanoma‐targeted chemo‐thermo‐immuno (CTI)‐therapy using N‐propionyl‐4‐S‐cysteaminylphenol‐magnetite nanoparticles elicits CTL response via heat shock protein‐peptide complex release

Melanogenesis substrate, N‐propionyl‐4‐S‐cysteaminylphenol (NPrCAP) is specifically taken up by melanoma cells and inhibits their growth by producing cytotxic free radicals. By taking advantage of this unique chemical agent, we have established melanoma‐targeting intracellular hyperthermia by conjugating NPrCAP with magnetite nanoparticles (NPrCAP/M) upon exposure to an alternating magnetic field (AMF). This treatment causes cytotoxic reaction as well as heat shock responses, leading to elicitation of antitumor immune response, which was proved by tumor rechallenge test and CTL induction. We found the level of heat shock protein 72 (Hsp72) to be increased in the cell lysate and culture supernatant after intracellular hyperthermia. Melanoma‐specific CD8⁺ T‐cell response to dendritic cells loaded with hyperthermia‐treated tumor lysate was enhanced when compared with non‐treated tumor lysate. When heat shock protein, particularly Hsp72, was immuno‐depleted from hyperthermia‐treated tumor cell lysate, specific CD8⁺ T‐cell response was abolished. Thus, it is suggested that antitumor immune response induced by hyperthermia using NPrCAP/M is derived from the release of HSP‐peptide complex from degraded tumor cells. Therefore, this chemo‐thermo‐immuno (CTI)‐therapy might be effective not only for primary melanoma but also for distant metastasis because of induction of systemic antimelanoma immune responses. (Cancer Sci 2010)     

Granulocyte‐colony‐stimulating factor enhances invasive potential of human head‐and‐neck‐carcinoma cell lines

Granulocyte‐colony‐stimulating factor (G‐CSF), a hematopoietic cytokine, regulates the proliferation and differentiation of granulocytic progenitor cells and functionally activated mature neutrophils. G‐CSF also affects non‐hematopoietic tumor cells by the binding of G‐CSF to its specific receptor (G‐CSFR) on the cells. In this study, we investigated the effect of G‐CSF on the invasive potential of head‐and‐neck carcinoma cells, and explored the intracellular events initiated by the binding of G‐CSF in tumor cells. In vitro treatment of head‐and‐neck‐carcinoma cell lines, IMC‐2, IMC‐3, KB, Ca9‐22, SCCKN and SCCTF, with recombinant G‐CSF (rG‐CSF) significantly augmented their invasive potential in dose‐ and time‐dependent manners. Among these cancer cells, IMC‐2, IMC‐3, KB and Ca9‐22 cells produced little G‐CSF, while large amounts of G‐CSF were produced by SCCKN and SCCTF cell lines. Anti‐G‐CSF antibody (Ab) abrogated the rG‐CSF‐enhanced invasiveness to the control level of that in untreated cancer cell lines. Immunocytochemical staining and Western blotting using anti‐G‐CSFR monoclonal antibody (MAb) revealed the expression of G‐CSFR on head‐and‐neck‐cancer cell lines exhibiting the enhancement of invasive activity by rG‐CSF. IMC‐2 cells, having the highest invasive ability among the cell lines used, showed augmentation of G‐CSFR expression on stimulation with rG‐CSF. Furthermore, stimulation of IMC‐2 cells with rG‐CSF induced rapid activation of tyrosine‐phosphorylated JAK1, suggesting that the G‐CSF signal may be transduced into the cells through G‐CSFR. Moreover, the gelatinolytic activity of IMC‐2 cells was enhanced by stimulation of rG‐CSF, and the enhanced invasiveness was inhibited on addition of the tissue inhibitors of metalloproteinases (TIMPs). These results suggest that exogenous rG‐CSF may increase the risk of metastasis and/or local recurrence in patients with G‐CSFR‐positive head‐and‐neck squamous‐cell carcinoma, via an invasive mechanism. Int. J. Cancer 80:78–84, 1999. © 1999 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     

Molecular Characterization of Basal‐Like and Non‐Basal‐Like Triple‐Negative Breast Cancer

Triple-negative (TN) and basal-like (BL) breast cancer definitions have been used interchangeably to identify breast cancers that lack expression of the hormone receptors and overexpression and/or amplification of HER2. However, both classifications show substantial discordance rates when compared to each other. Here, we molecularly characterize TN tumors and BL tumors, comparing and contrasting the results in terms of common patterns and distinct patterns for each. In total, when testing 412 TN and 473 BL tumors, 21.4% and 31.5% were identified as non-BL and non-TN, respectively. TN tumors identified as luminal or HER2-enriched (HER2E) showed undistinguishable overall gene expression profiles when compared versus luminal or HER2E tumors that were not TN. Similar findings were observed within BL tumors regardless of their TN status, which suggests that molecular subtype is preserved regardless of individual marker results. Interestingly, most TN tumors identified as HER2E showed low HER2 expression and lacked HER2 amplification, despite the similar overall gene expression profiles to HER2E tumors that were clinically HER2-positive. Lastly, additional genomic classifications were examined within TN and BL cancers, most of which were highly concordant with tumor intrinsic subtype. These results suggest that future clinical trials focused on TN disease should consider stratifying patients based upon BL versus non-BL gene expression profiles, which appears to be the main biological difference seen in patients with TN breast cancer.     

miR‐135b‐ and miR‐146b‐dependent silencing of calcium‐sensing receptor expression in colorectal tumors

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = ?0.684, p < 0.001 and miR‐146b: r = ?0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.     

Population‐based 10‐year oncologic outcomes after low‐dose‐rate brachytherapy for low‐risk and intermediate‐risk prostate cancer

BACKGROUND.

The objective of this study was to report the rates of disease‐free survival (DFS), cause‐specific survival (CSS), and overall survival after low‐dose‐rate (LDR) prostate brachytherapy (PB).

METHODS.

Data from 1006 consecutive patients with prostate cancer who received LDR‐PB and underwent implantation on or before October 23, 2003 were extracted from a prospective database on November 11, 2011. The selected patients had low‐risk (58%) or intermediate‐risk (42%) disease according to National Comprehensive Cancer Network criteria. The Phoenix threshold was used to define biochemical relapse. Sixty‐five percent of patients received 3 months of neoadjuvant androgen‐deprivation therapy (ADT) and 3 months of concomitant ADT. Univariate and multivariate analyses are reported in relation to patient, tumor, and treatment variables.

RESULTS.

The median follow‐up was 7.5 years. By using Fine and Gray competing risks analysis, the 5‐year and 10‐year actuarial DFS rates were 96.7% (95% confidence interval, 95.2%‐97.7%) and 94.1% (95% confidence interval, 92%‐95.6%), respectively. When applied to the whole cohort, none of the usual prognostic variables, including dose metrics, were correlated with DFS. However, in both univariate and multivariate models, increasing dose was the only covariate that correlated with improved DFS for the subset of men (N = 348) who did not receive ADT (P = .043). The actuarial 10‐year CSS rate was 99.1% (95% confidence interval, 97.3%‐99.7%). The overall survival rate was 93.8% at 5 years (95% confidence interval, 92%‐95.1%) and 83.5% at 10 years (95% confidence interval, 79.8%‐86.6%). Only age at implantation (P = .0001) was correlated with overall survival in multivariate analysis.

CONCLUSIONS.

In a consecutive cohort of 1006 men with National Comprehensive Cancer Network low‐risk and intermediate‐risk prostate cancer, the actuarial rate of recurrent disease after LDR‐PB was approximately 3% at 5 years and 6% at 10 years. Cancer 2013. © 2012 American Cancer Society.     

Prognostic value of health‐related quality‐of‐life parameters in early‐stage breast cancer: an 8‐year follow‐up study

Objective: The purpose was to investigate whether self‐reported health‐related quality‐of‐life (HRQOL) parameters at time of diagnosis and/or 1‐year follow‐up are prognostic for disease‐free survival (DFS) in early‐stage breast cancer patients. Methods: Data from 195 women, diagnosed with early‐stage breast cancer, who had filled in the EORTC QLQ‐C30 and the Hospital Anxiety and Depression Scale (HADS) at time of diagnosis and 1 year after surgery, were analyzed. Results: After a median follow‐up of 8.2 years (range 0.09–9.45), 27 (14.1%) deaths and 22 (11.5%) recurrences were observed. Using Cox multivariate regression analysis, appetite loss reported 1‐year following surgery (HR 2.92, 95% CI 1.50–5.66), p=0.002) was significantly predictive for shorter DFS, even after controlling for age and depression. None of the clinical or biological prognostic factors was found to have a confounding effect. Conclusion: The findings indicate that loss of appetite probably is of prognostic value in addition to well‐recognized clinical and biological data, in early‐stage breast cancer. Copyright © 2010 John Wiley & Sons, Ltd.     

Nab‐paclitaxel interrupts cancer‐stromal interaction through C‐X‐C motif chemokine 10‐mediated interleukin‐6 downregulation in vitro

Cancer‐associated fibroblasts (CAF), derived from stroma of cancer tissues, interact with cancer cells and play an important role in cancer initiation, growth, and metastasis. Nab‐paclitaxel (nab‐PTX) is a 130 nm albumin‐binding paclitaxel and recommended for many types of cancer chemotherapy. The nab‐PTX stromal‐disrupting effect during pancreatic cancer treatment has been reported. The aim of the present study was to determine the role of nab‐PTX in cancer cells and CAF interaction. Cancer cells (MIA PaCa‐2 and Panc‐1) were cocultured with CAF or treated with CAF conditioned medium, after which their migration and invasion ability, epithelial‐mesenchymal transition (EMT)‐related marker expression and C‐X‐C motif chemokine 10 (CXCL10) expression and secretion were detected. Nab‐PTX treatment was carried out during the coculture system or during preparation of CAF conditioned medium. Then cancer cell migration and invasion ability, EMT‐related marker expression, CXCL10 expression and secretion, and interleukin‐6 (IL‐6) expression and secretion by CAF were checked After coculture with CAF, migration and invasion ability of cancer cells increased. CAF also downregulated E‐cadherin and upregulated N‐cadherin and vimentin expression in cancer cells. During coculture or stimulation with cancer cell‐cultured medium, CAF significantly increased IL‐6 expression and secretion. However, nab‐PTX in the coculture system canceled CAF‐induced migration and invasion promotion and EMT‐related gene changes. Moreover, nab‐PTX increased CXCL10 expression of cancer cells which blocked CAF IL‐6 expression and secretion. Nab‐PTX treatment could increase CXCL10 expression of cancer cells which blocks CAF cancer cell migration and invasion‐promoting effect by inhibiting IL‐6 expression.     

Krüppel‐like factor 4 promotes c‐Met amplification‐mediated gefitinib resistance in non‐small‐cell lung cancer

Gefitinib has been widely used in the first‐line treatment of advanced EGFR‐mutated non‐small‐cell lung cancer (NSCLC). However, many NSCLC patients will acquire resistance to gefitinib after 9‐14 months of treatment. This study revealed that Krüppel‐like factor 4 (KLF4) contributes to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells. We observed that KLF4 was overexpressed in c‐Met‐overexpressing NSCLC cells and tissues. Knockdown of KLF4 increased tumorigenic properties in gefitinib‐resistant NSCLC cell lines without c‐Met overexpression, but it reduced tumorigenic properties and increased gefitinib sensitivity in gefitinib‐resistant NSCLC cells with c‐Met overexpression, whereas overexpression of KLF4 reduced gefitinib sensitivity in gefitinib‐sensitive NSCLC cells. Furthermore, Western blot analysis revealed that KLF4 contributed to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells by inhibiting the expression of apoptosis‐related proteins under gefitinib treatment and activating the c‐Met/Akt signaling pathway by decreasing the inhibition of β‐catenin on phosphorylation of c‐Met to prevent blockade by gefitinib. In summary, this study's results suggest that KLF4 is a promising candidate molecular target for both prevention and therapy of NSCLC with c‐Met overexpression.     

Enhanced tumorigenesis of forestomach tumors induced by N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine in rats with hypoinsulinemic diabetes

Hyperinsulinemia and hyperglycemia in prediabetic and diabetic patients are thought to increase the risk of developing neoplasms because insulin is a growth factor with pre‐eminent metabolic but also mitogenic effects. To determine the effect of hypoinsulinemic diabetic conditions on carcinogenesis, we examined N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG)‐induced forestomach carcinogenesis in hypoinsulinemic diabetic WBN/Kob rats aged about 45 weeks (DM) compared with non‐diabetic younger WBN/Kob rats (C1), non‐diabetic Wistar rats age‐matched to DM (C2), and non‐diabetic Wistar rats age‐matched to C1 (C3). All rats were treated with MNNG by gavage and were killed at 40 weeks after dosing. Various‐sized tumors were disseminated throughout the forestomach of all rats, and the ratio of the area of tumors to the whole forestomach area was 23.3% in the DM group and was higher than in the C1–3 (4.2–14.3%) groups. The incidence of carcinoma was much higher in the DM group (36.8%) than in the C1–3 (7.1–16.7%) groups, and the incidence of papilloma was also significantly higher in the DM group (84.2%) than in the C1–3 (28.5–50.0%) groups. The average thickness of the squamous epithelium in the non‐neoplastic mucosa was significantly greater in the DM group (50.8 μm) than in the C1–3 (29.6–37.9 μm) groups. Immunohistochemically, the Ki‐67‐positive index in the non‐tumorous mucosa of the DM group (42.0%) was significantly higher than that of the C1–3 groups (18.8–33.3%). These results suggest that prolonged hyperglycemic conditions without hyperinsulinemia enhance tumorigenesis of MNNG‐induced tumors by enhanced proliferative activity of the squamous epithelium in the rat forestomach. (Cancer Sci 2010)     

Cervical cancer cell‐derived interleukin‐6 impairs CCR7‐dependent migration of MMP‐9‐expressing dendritic cells

Cervical carcinogenesis is a consequence of persistent infection with high‐risk human papillomaviruses (HPVs). Recent studies indicate that HPV‐transformed cells actively instruct their microenvironment to promote carcinogenesis. Here, we demonstrate that cervical cancer cells activate monocytes to produce their own CCL2 for further monocyte recruitment and reprogram their function during differentiation and maturation to dendritic cells (DCs). Our data show that cervical cancer cells suppress the induction of the chemokine receptor CCR7 in phenotypically mature DCs and impair their migration toward a lymph node homing chemokine, required to initiate adaptive immune responses. We confirmed the presence of CD83⁺CCR7^low DCs in cancer biopsies. The second factor essential for DC migration, matrix‐metalloproteinase MMP‐9, which also has vasculogenic and protumorigenic properties, is not suppressed but upregulated in immature as well as mature DCs. We identified interleukin‐6 (IL‐6) as a crucial cervical cancer cell‐derived mediator and nuclear factor kappaB (NF‐κB) as the central signaling pathway targeted in DCs. Anti‐IL‐6 antibodies reverted not only NF‐κB inhibition and restored CCR7‐dependent migration but also blocked MMP‐9 induction. This is the first report demonstrating the dissociation of CCR7 and MMP‐9 expression in phenotypically mature CD83⁺ DCs by cancer cells. Our results show that cervical cancer cells actively shape the local microenvironment. They induce the accumulation of myeloid cells and skew their function from immune activation to local production of protumorigenic MMP‐9. Neutralizing anti‐IL‐6 antibodies can counteract this functional dysbalance and should therefore be considered for adjuvant cervical cancer therapy.     

c‐Src promotes tumor progression through downregulation of microRNA‐129‐1‐3p

MicroRNAs (miRNAs) fine‐tune cellular signaling by regulating expression of signaling proteins, and aberrant expression of miRNAs is observed in many cancers. The tyrosine kinase c‐Src is upregulated in various human cancers, but the molecular mechanisms underlying c‐Src‐mediated tumor progression remain unclear. In previous investigations of miRNA‐mediated control of c‐Src‐related oncogenic pathways, we identified miRNAs that were downregulated in association with c‐Src transformation and uncovered the signaling networks by predicting their target genes, which might act cooperatively to control tumor progression. Here, to further elucidate the process of cell transformation driven by c‐Src, we analyzed the expression profiles of miRNAs in a doxycycline‐inducible Src expression system. We found that miRNA (miR)‐129‐1‐3p was downregulated in the early phase of c‐Src‐induced cell transformation, and that reexpression of miR‐129‐1‐3p disrupted c‐Src‐induced cell transformation. In addition, miR‐129‐1‐3p downregulation was tightly associated with tumor progression in human colon cancer cells/tissues. Expression of miR‐129‐1‐3p in human colon cancer cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasion. We also identified c‐Src and its critical substrate Fer, and c‐Yes, a member of the Src family of kinases, as novel targets of miR‐129‐1‐3p. Furthermore, we found that miR‐129‐1‐3p‐mediated regulation of c‐Src/Fer and c‐Yes is important for controlling cell adhesion and invasion. Downregulation of miR‐129‐1‐3p by early activation of c‐Src increases expression of these target genes and synergistically promotes c‐Src‐related oncogenic signaling. Thus, c‐Src‐miR‐129‐1‐3p circuits serve as critical triggers for tumor progression in many human cancers that harbor upregulation of c‐Src.