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.     

O‐phospho‐L‐tyrosine protects TP53 wild‐type cells against ionizing radiation

O‐phospho‐L‐tyrosine (P‐Tyr) has been reported previously to inhibit growth of several cancer cell lines at mM concentrations. In the present study, we investigated the effect of this compound on tumor cells and normal cells in combination with radiation exposure. It could be demonstrated for the first time that P‐Tyr at μM concentrations protects TP53 wild‐type cells against ionizing radiation (SF4 minus BBI = 0.28, SF4 plus BBI = 0.45). On the contrary, human transformed or tumor cell lines characterized by mutated or functional inactivated TP53 were not altered or increased in their radiation sensitivity (SF4 minus BBI = 0.32, SF4 plus BBI = 0.22). Treatment of wild‐type TP53 cells with P‐Tyr induced stabilization of TP53 within 3 and 16 hours and a subsequent increase in CDKN1A expression after treatment. Consequently, a 16‐hours pretreatment of cells with P‐Tyr led to a significant radioprotective effect. This was not observed in cell lines with mutated TP53, which shows no radioprotection by P‐Tyr. Thus, the present data suggest that P‐Tyr‐mediated radioprotection is dependent on preirradiation stabilization of TP53. The results indicate that P‐Tyr is a radioprotective agent that can potentially be very useful and easy to deliver for radiation protection in general and especially in radiation therapy of TP53‐mutated tumors. © 2002 Wiley‐Liss, Inc.     

MiR‐483‐5p and miR‐139‐5p promote aggressiveness by targeting N‐myc downstream‐regulated gene family members in adrenocortical cancer

Adrenocortical carcinoma (ACC) is a tumor with poor prognosis in which overexpression of a panel of microRNAs has been associated with malignancy but a very limited number of investigations on their role in ACC pathogenesis have been conducted. We examined the involvement of miR‐483‐5p and miR‐139‐5p in adrenocortical cancer aggressiveness. Using bioinformatics predictions and mRNA/miRNA expression profiles, we performed an integrated analysis to identify inversely correlated miRNA–mRNA pairs in ACC. We identified N‐myc downstream‐regulated gene family members 2 and 4 (NDRG2 and NDRG4) as targets of miR‐483‐5p and miR‐139‐5p, respectively. NDRG2 and NDRG4 expressions were inversely correlated respectively with miR‐483‐5p and miR‐139‐5p levels in aggressive ACC samples from two independent cohorts of 20 and 44 ACC. Moreover, upregulation of miR‐139‐5p and downregulation of NDRG4 demonstrated a striking prognostic value. A direct interaction between miR‐483‐5p or miR‐139‐5p and their targets was demonstrated in reporter assays. Downregulation of miR‐483‐5p or miR‐139‐5p in the ACC cell lines NCI‐H295R and SW13 increased NDRG2 or NDRG4 mRNA and protein expression, compromised adrenocortical cancer cell invasiveness and anchorage‐independent growth. MiR‐483‐5p or miR‐139‐5p overexpression and NDRG2 or NDRG4 inhibition produce similar changes, which are rescued by NDRG2 or NDRG4 ectopic expression. We established that key factors mediating epithelial‐to‐mesenchymal transition are downstream effectors of miR‐483‐5p/NDRG2 and miR‐139‐5p/NDRG4 pathways. Collectively, our data show for the first time that miR‐483‐5p/NDRG2 and miR‐139‐5p/NDRG4 axes promote ACC aggressiveness, with potential implications for prognosis and therapeutic interventions in adrenocortical malignancies.     

Health‐related quality‐of‐life in patients with head‐and‐neck cancer in Sri Lanka: psychometric properties of the ‘Sinhala’ version of the EORTC QLQ‐H&N35

Objective: To translate and validate the ‘Sinhala’ language version of the European Organization for Research and Treatment of Cancer head‐and‐neck cancer‐specific health‐related quality‐of‐life questionnaire module, the QLQ‐H&N35, for use in Sri Lanka. Methods: Psychometric testing assessed the hypothesized scale structure, scale reliability, construct validity and acceptability of the translated version of the QLQ‐H&N35 in a consecutive series of 196 newly diagnosed head‐and‐neck cancer patients, recruited from tertiary‐care oncology treatment centres in Sri Lanka. Results: Compliance was high (97.5%), although nearly 40% of patients required assistance with completion of the questionnaire. Twenty‐four sexually inactive patients declined to answer one or both items of the sexuality scale. Multi‐trait scaling confirmed the overall scale structure, with good item‐convergent (100%) and ‐discriminant (93.8%) validity, and scaling success (86.8%) rates. Cronbach's alpha coefficients exceeded 0.70 for all scales, except problems with sexuality (0.60) and problems with senses (0.61), which also evidenced a lower scaling success rate (50%). Confirmation of construct validity included satisfactory results for inter‐scale correlations and known‐groups comparisons for most scales; most correlations were statistically significant (p<0.01), with conceptually related scales showing relatively higher correlation. Most scale scores were able to discriminate clearly between pre‐ and current treatment patients. Conclusions: Results of the study provide strong support for the psychometric robustness of the ‘Sinhala’ version of the QLQ‐H&N35. It may be advisable to interpret the two items assessing sensory problems separately, and to elicit information on sexuality from only those who are sexually active. Copyright © 2009 John Wiley & Sons, Ltd.     

Classifying B‐cell non‐Hodgkin lymphoma by using MIB‐1 proliferative index in fine‐needle aspirates

BACKGROUND:

MIB‐1 proliferation index (PI) has proven helpful for diagnosis and prognosis in non‐Hodgkin lymphomas (NHLs). However, validated cutoff values for use in fine‐needle aspiration (FNA) samples are not available. We investigated MIB‐1 immunocytochemistry as an ancillary technique for stratifying NHL and attempted to establish PI cutpoints in cytologic samples.

METHODS:

B‐cell NHL FNA cases with available cytospins (CS) MIB‐1 immunocytochemistry results were included. Demographic, molecular, immunophenotyping and MIB‐1 PI data were collected from cytologic reports. Cases were subtyped according to the current World Health Organization classification and separated into indolent, aggressive, and highly aggressive groups. Statistical analysis was performed with pairwise Wilcoxon rank sum test and linear discriminant analysis to suggest appropriate PI cutpoints.

RESULTS:

Ninety‐one NHL cases were subdivided in 56 (61.5%) indolent, 30 (33%) aggressive, and 5 (5.5%) highly aggressive lymphomas. The 3 groups had significantly different MIB‐1 PIs from each other. Cutpoints were established for separating indolent (<38%), aggressive (≥38% to ≤80.1%) and highly aggressive (>80.1%). The groups were adequately predicted in 76 cases (83.5%) using the cutpoints and 15 cases showed discrepant PIs.

CONCLUSIONS:

MIB‐1 immunohistochemistry on CS can help to stratify B‐cell NHL and showed a significant increase in PI with tumor aggressiveness. Six misclassified cases had PIs close to the cutpoints. Discrepant MIB‐1 PIs were related to dilution of positive cells by non‐neoplastic lymphocytes and to the overlapping continuum of features between diffuse large B‐cell lymphoma and Burkitt lymphoma. Validation of our approach in an unrelated, prospective dataset is required. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

Ikaros is a critical target during simultaneous exposure to X‐rays and N‐ethyl‐N‐nitrosourea in mouse T‐cell lymphomagenesis

Cancer risk associated with radiation exposure is considered the result of concurrent exposure to other natural and manmade carcinogens. Available data on the molecular characteristics of cancer after simultaneous exposure to radiation and chemicals are insufficient. In our study, we used a mouse thymic lymphoma (TL) model that was synergistically induced by simultaneous exposure to X‐rays and N‐ethyl‐N‐nitrosourea (ENU) at subcarcinogenic doses and analyzed the mutation frequency and spectrum of the TL‐associated genes Ikaros, Notch1, p53 and Kras. We found that the point mutation frequency in Ikaros was significantly increased to 47% for simultaneous exposure compared to 13 and 0% for X‐ray and ENU exposure alone, respectively. These mutations were mostly G:C > A:T at non‐CpG sites and T:A > C:G, both of which are characteristic of ENU mutagenesis. About half of the point mutations were accompanied by loss of heterozygosity (LOH), typical of X‐irradiation. The remaining half did not include LOH, which suggests that they were dominant‐negative mutations. In Notch1, the frequency of abnormalities was high (>58%) regardless of the treatment, suggesting that Notch1 aberration may be important for T‐cell lymphomagenesis. The p53 and Kras mutation frequencies were low for all treatments (<23%). Importantly, the frequency of TLs containing mutations in multiple genes, especially both Ikaros and Notch1, increased after simultaneous exposure. Thus, after simultaneous exposure, Ikaros is a critical target and is inactivated by ENU‐induced point mutations and/or X‐ray‐induced LOH in T‐cell lymphomagenesis. Furthermore, concomitant alterations of multiple tumor‐associated genes may contribute to enhanced lymphomagenesis after simultaneous exposure.     

μ‐Calpain regulates caspase‐dependent and apoptosis inducing factor‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis

Cisplatin, an effective anticancer agent, can induce tumor cell apoptosis via caspase‐dependent and‐independent pathways. However, the precise mechanism that regulates the pathways remains unclear. In this study, we showed that μ‐calpain mediated both caspase‐dependent and‐independent pathways during cisplatin‐induced apoptosis in human lung adenocarcinoma cells. After cisplatin treatment, calpain activation, as measured by a fluorescent substrate, was an early event, taking place well before apoptosis inducing factor (AIF) release and caspase‐9/‐3 activation. Confocal imaging of cells transfected with AIF‐GFP demonstrated that AIF release occurred about 9 hr after cisplatin treatment. The increase of μ‐calpain activity proved to be a crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples suppressed the endogenous μ‐calpain expression level, as well as cotreated with the calpain inhibitors, calpeptin and PD150606. Inhibition of μ‐calpain not only significantly reduced caspase‐9/‐3 activities but also completely blocked AIF redistribution. Our study also showed that endogenous mitochondrial μ‐calpain could directly induce the truncation and release of AIF, while caspases and cathepsins were not necessary for this process. In conclusion, the study demonstrated that activation of μ‐calpain played an essential role in regulating both caspase‐dependent and AIF‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis. © 2009 UICC     

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.     

Early 18F‐2‐fluoro‐2‐deoxy‐d‐glucose positron emission tomography may identify a subset of patients with estrogen receptor‐positive breast cancer who will not respond optimally to preoperative chemotherapy

BACKGROUND:

A pathologic complete response (pCR) and minimal residual disease (pMRD) after preoperative chemotherapy (PCT) for early stage or locally advanced breast cancer (BC) correlates with a good prognosis.

METHODS:

Patients who received from 6 to 8 cycles of PCT for BC were monitored by ¹⁸F‐2‐fluoro‐2‐deoxy‐D‐glucose positron emission tomography (¹⁸F‐FDG‐PET), and the maximal standardized uptake value (SUVmax) was calculated at baseline, after 2 cycles, after 4 cycles, and at the end of PCT. SUVmax percentage changes (Δ‐SUV) were compared with the pathologic response rate. Patients who had a pCR or pMRD in the tumor and an absence of cancer cells in ipsilateral axillary lymph nodes were defined as having obtained an optimal pathologic response (pR), whereas all the other conditions were classified as a pathologic nonresponse (pNR).

RESULTS:

Of 34 patients, 7 (21%) achieved a pR (3 patients had a pCR, and 4 patients had pMRD). After the second cycle, the Δ‐SUV threshold with optimal negative predictive value to predict a pR was 50%. Twenty‐six patients (76%) had a Δ‐SUV >50%, including all 7 patients who had a pR and 19 patients who had a pNR. Conversely, all 8 patients who had a Δ‐SUV ≤50% had a pNR. All 8 of those patients had estrogen recepetor‐positive tumors.

CONCLUSIONS:

Early evaluation of metabolic response by ¹⁸F‐FDG‐PET during PCT was able to identify 30% of patients, all with estrogen receptor‐positive tumors, who would not obtain pR after completion of chemotherapy program. Cancer 2010. © 2010 American Cancer Society.     

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     

TNF‐α enhances TGF‐β‐induced endothelial‐to‐mesenchymal transition via TGF‐β signal augmentation

The tumor microenvironment (TME) consists of various components including cancer cells, tumor vessels, cancer‐associated fibroblasts (CAFs), and inflammatory cells. These components interact with each other via various cytokines, which often induce tumor progression. Thus, a greater understanding of TME networks is crucial for the development of novel cancer therapies. Many cancer types express high levels of TGF‐β, which induces endothelial‐to‐mesenchymal transition (EndMT), leading to formation of CAFs. Although we previously reported that CAFs derived from EndMT promoted tumor formation, the molecular mechanisms underlying these interactions remain to be elucidated. Furthermore, tumor‐infiltrating inflammatory cells secrete various cytokines, including TNF‐α. However, the role of TNF‐α in TGF‐β‐induced EndMT has not been fully elucidated. Therefore, this study examined the effect of TNF‐α on TGF‐β‐induced EndMT in human endothelial cells (ECs). Various types of human ECs underwent EndMT in response to TGF‐β and TNF‐α, which was accompanied by increased and decreased expression of mesenchymal cell and EC markers, respectively. In addition, treatment of ECs with TGF‐β and TNF‐α exhibited sustained activation of Smad2/3 signals, which was presumably induced by elevated expression of TGF‐β type I receptor, TGF‐β2, activin A, and integrin αv, suggesting that TNF‐α enhanced TGF‐β‐induced EndMT by augmenting TGF‐β family signals. Furthermore, oral squamous cell carcinoma‐derived cells underwent epithelial‐to‐mesenchymal transition (EMT) in response to humoral factors produced by TGF‐β and TNF‐α‐cultured ECs. This EndMT‐driven EMT was blocked by inhibiting the action of TGF‐βs. Collectively, our findings suggest that TNF‐α enhances TGF‐β‐dependent EndMT, which contributes to tumor progression.     

Atorvastatin inhibits pancreatic carcinogenesis and increases survival in LSL‐KrasG12D‐LSL‐Trp53R172H‐Pdx1‐Cre mice

There are several studies supporting the role of HMG‐CoA reductase inhibitors such as atorvastatin against carcinogenesis, in which inhibiting the generation of prenyl intermediates involved in protein prenylation plays the crucial role. Mutation of Kras gene is the most common genetic alteration in pancreatic cancer and the Ras protein requires prenylation for its membrane localization and activity. In the present study, the effectiveness of atorvastatin against pancreatic carcinogenesis and its effect on protein prenylation were determined using the LSL‐Kras^G12D‐LSL‐Trp53^R172H‐Pdx1‐Cre mouse model (called Pan^kras/p53 mice). Five‐week‐old Pan^kras/p53 mice were fed either an AIN93M diet or a diet supplemented with 100 ppm atorvastatin. Kaplan–Meier survival analysis with Log‐Rank test revealed a significant increase in survival in mice fed 100 ppm atorvastatin (171.9 ± 6.2 d) compared to the control mice (144.9 ± 8.4 d, P < 0.05). Histologic and immunohistochemical analysis showed that atorvastatin treatment resulted in a significant reduction in tumor volume and Ki‐67‐labeled cell proliferation. Mechanistic studies on primary pancreatic tumors and the cultured murine pancreatic carcinoma cells revealed that atorvastatin inhibited prenylation in several key proteins, including Kras protein and its activities, and similar effect was observed in pancreatic carcinoma cells treated with farnesyltransferase inhibitor R115777. Microarray assay on the global gene expression profile demonstrated that a total of 132 genes were significantly modulated by atorvastatin; and Waf1p21, cyp51A1, and soluble epoxide hydrolase were crucial atorvastatin‐targeted genes which involve in inflammation and carcinogenesis. This study indicates that atorvastatin has the potential to serve as a chemopreventive agent against pancreatic carcinogenesis. © 2012 Wiley Periodicals, Inc.     

BOK displays cell death‐independent tumor suppressor activity in non‐small‐cell lung carcinoma

As the genomic region containing the Bcl‐2‐related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non‐small‐cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node‐positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFβ‐induced migration and epithelial‐to‐mesenchymal transition (EMT) in lung adenocarcinoma‐derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low‐BOK‐expressing tumors might favor the overall survival of NSCLC patients.     

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.