Risk factors for progressive sarcopenia 6 months after complete resection of lung cancer: what can thoracic surgeons do against sarcopenia?

BACKGROUND: Our previous report described how postoperative progression of sarcopenia predicted long-term prognosis after complete resection of non-small cell lung cancer (NSCLC) in heavy smokers. However, there are currently no effective means to treat progressive sarcopenia. In this study, we aimed to confirm our previous findings in a larger population and to identify factors associated with postoperative progression of sarcopenia to propose possible preventative measures. METHODS: This retrospective study analyzed the data of 1,095 patients who underwent curative lobar resection for NSCLC at Kanagawa Cancer Center. We divided patients into four groups according to sex and Brinkman index (BI) above or below 600. Six-month postoperative changes in the skeletal muscle index (SMI) were calculated and associations between clinicopathological factors including changes in SMI and mortality from postoperative 6 months were examined. Only in groups in which postoperative depletion of SMI was shown to be associated with the prognosis, we identified clinicopathological factors associated with depletive SMI. RESULTS: The overall survival rates of 1,095 patients were 89.8% and 82.5% at 3 and 5 years, respectively. The median 6-month change in SMI was -3.4% (range, -22.3% to +17.9%). Multivariate analysis revealed that poor prognosis was independently predicted by a large reduction in the SMI (cut-off value: -10%) in males with a BI ≥600. In 391 heavy-smoking males, factors associated with a postoperative change in SMI ≤-10% were history of other cancers (including gastric cancer) low forced expiratory volume in one second (FEV 1.0, cut-off value: 1,870 mL), and prolonged operation time (cut-off value: 200 minutes). CONCLUSIONS: Perioperative measures to prevent postoperative sarcopenia are appropriate for heavy smokers. We obtained some clues regarding countermeasures, one of which may be avoiding long-time operation. Further studies including clinical trials to assess perioperative anti-sarcopenia treatments, are needed.

Prognostic value of volumetric FDG PET/CT parameters in patients with oral tongue squamous cell carcinoma who were treated by superselective intra-arterial chemoradiotherapy.

PURPOSE: This study aimed to evaluate the predictive and prognostic value of FDG PET/CT-based volumetric parameters in patients with oral tongue squamous cell carcinoma (OTSCC) treated by superselective intra-arterial chemoradiotherapy (IA-CRT). METHODS: We conducted a retrospective study including 33 patients with biopsy-proven OTSCC between May 2007 and February 2016. All of the patients were treated by IA-CRT. Pretreatment SUVmax and metabolic tumor volume (MTV) of the primary tumor were measured. The SUV thresholds of 2.5 and 5.0 were used. Progression-free survival (PFS) and overall survival (OS) were chosen as endpoints to evaluate prognosis. Univariate and multivariate analyses were performed to assess the potential independent effect of FDG PET/CT parameters. RESULTS: The median follow-up for surviving patients was 40.7 months (range 6.0-107.5 months). In univariate and multivariate analyses, SUVmax and MTV (5.0) were independent prognostic factors for PFS. In univariate analysis, SUVmax failed to predict OS. MTV (5.0) was a significant prognostic factor for OS, but multivariate analysis failed to show statistical independence because it could not exclude the possibility of an artifact due to N stage. CONCLUSIONS: FDG PET/CT-based volumetric parameters may be significant prognostic markers for survival of patients with OTSCC who are treated by IA-CRT.

Long Noncoding RNA JHDM1D-AS1 Promotes Tumor Growth by Regulating Angiogenesis in Response to Nutrient Starvation.

Long noncoding RNAs play a pivotal role in tumor progression, but their role in cancer cells in the nutrient-starved tumor microenvironment remains unknown. Here, we show that a nutrient starvation-responsive long noncoding RNA, JHDM1D antisense 1 (JHDM1D-AS1), promotes tumorigenesis by regulating angiogenesis in response to nutrient starvation. Expression of JHDM1D-AS1 was increased in cancer cells. In addition, expression of JHDM1D-AS1 was increased in clinical tumor samples compared to that in normal tissue. Stable expression of JHDM1D-AS1 in human pancreatic cancer (PANC-1 and AsPC-1) cells promoted cell growth in vitro Remarkably, these JHDM1D-AS1-expressing cells showed a significant increase in tumor growth in vivo that was associated with increased formation of CD31+ blood vessels and elevated infiltration of CD11b+ macrophage lineage cells into tumor tissues. Genome-wide analysis of tumor xenografts revealed that expression of genes for tumor-derived angiogenic factors such as hHGF and hFGF1 concomitant with host-derived inflammation-responsive genes such as mMmp3, mMmp9, mS100a8, and mS100a9 was increased in tumor xenografts of JHDM1D-AS1-expressing pancreatic cancer cells, leading to a poor prognosis. Our results provide evidence that increased JHDM1D-AS1 expression under nutrient starvation accelerates tumor growth by upregulating angiogenesis, thus laying the foundation for improved therapeutic strategies.

Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.

Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification.

Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.

tRNase-Z(L)-utilizing efficacious (TRUE) gene silencing is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the property of tRNase Z(L) that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA). To search for novel potential therapeutic sgRNAs for hematological malignancies, we screened a library composed of 156 sgRNAs, and found that 20 sgRNAs can efficiently induce apoptosis in leukemia and/or myeloma cells. Furthermore, we demonstrated that 4 of the 20 sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models.

The anti-overactive bladder activity of KW-7158 is mediated by blocking equilibrative nucleoside transporter-1.

KW-7158 is a novel therapeutic candidate for treating overactive bladder (OAB) with a unique mode of action: suppression of sensory afferent nerves. However, the molecular target of this compound remains unknown. We herein report the identification of the KW-7158 target to be equilibrative nucleoside transporter-1 (ENT1). A membrane protein expression library of ca. 7000 genes was expressed in a dorsal root ganglion cell line, which we had previously generated, and subjected to screening for binding with a fluorescent derivative that retains high binding activity to the target. The screening revealed that only cells transfected with an ENT1 expression vector exhibited significant binding. We next performed [(3)H]KW-7158 binding experiments and an adenosine influx assay and found that KW-7158 binds to and inhibits ENT1. To further demonstrate the pharmacological relevance, we evaluated other known ENT1 inhibitors (nitrobenzylthioinosine, dipyridamole, draflazine) in an in vitro bladder strip contraction assay and the rat spinal cord injury OAB model. We found that all of the inhibitors exhibited anti-OAB activities, of which the potencies were comparable to that of adenosine influx inhibition in vitro. These studies demonstrated that the pharmacological target of KW-7158 is ENT1, at least in the rat OAB model. Our results will aid understanding of the precise mechanism of action of this drug and may also shed new light on the use of the adenosine pathway for the treatment of OAB.

miR-142-3p enhances FcεRI-mediated degranulation in mast cells.

Mast cells are immune cells derived from hematopoietic progenitors. When they are activated by stimuli, they immediately release granule-associated mediators, leading to allergic inflammation. Several factors controlling mediator release have been identified; however, little is known whether microRNAs (miRNAs) are involved in this process. miRNAs are a small class of non-coding RNAs that negatively regulate gene expression. In this study, we investigated the relationship between miRNAs and degranulation in LAD2 cells, a human mast cell line. We demonstrated that silencing of Dicer, a key enzyme of miRNA biogenesis, attenuates degranulation, indicating that miRNAs are involved in mast cell degranulation. We furthermore discovered that the overexpression of miR-142-3p enhances FcεRI-mediated degranulation and that miR-142-3p rescues the reduction of degranulation by silencing Dicer. Similar effects were observed in bone marrow-derived mast cells obtained miR-142-3p-deficient mice. Our studies suggest that miR-142-3p is a potential therapeutic target in pathological conditions caused by mast cells, such as mastocytosis and allergies.

Gain-of-function microRNA screens identify miR-193a regulating proliferation and apoptosis in epithelial ovarian cancer cells.

MicroRNAs (miRNAs) are a small class of non­coding RNAs that negatively regulate gene expression, and are considered as new therapeutic targets for treating cancer. In this study, we performed a gain-of-function screen using miRNA mimic library (319 miRNA species) to identify those affecting cell proliferation in human epithelial ovarian cancer cells (A2780). We discovered a number of miRNAs that increased or decreased the cell viability of A2780 cells. Pro-proliferative and anti-proliferative miRNAs include oncogenic miR-372 and miR-373, and tumor suppressive miR-124a, miR-7, miR-192 and miR-193a, respectively. We found that overexpression of miR-124a, miR-192, miR-193a and miR­193b inhibited BrdU incorporation in A2780 cells, indicating that these miRNAs affected the cell cycle. Overexpression of miR­193a and miR-193b induced an activation of caspase 3/7, and resulted in apoptotic cell death in A2780 cells. A genome­wide gene expression analysis with miR-193a-transfected A2780 cells led to identification of ARHGAP19, CCND1, ERBB4, KRAS and MCL1 as potential miR-193a targets. We demonstrated that miR-193a decreased the amount of MCL1 protein by binding 3'UTR of its mRNA. Our study suggests the potential of miRNA screens to discover miRNAs as therapeutic tools to treat ovarian cancer.

Induction of apoptosis of leukemic cells by TRUE gene silencing using small guide RNAs targeting the WT1 mRNA.

TRUE gene silencing is a technology to eliminate specific cellular RNAs by using tRNase Z(L) and small guide RNA (sgRNA). Here we investigated how WT1-mRNA-targeting sgRNAs affect leukemic cells. We showed that sgRNA can be easily taken up by cells without any transfection reagents, and that the naked sgRNAs targeting the WT1 mRNA can reduce its mRNA levels and WT1 protein amounts in the WT1-expressing leukemic cells. Concomitantly, these sgRNAs efficiently induced apoptosis in these cells but not in WT1-nonexpressing cells. We also demonstrated that the reduction in the WT1 mRNA level is caused by its cleavage by tRNase Z(L).

A prospective validation study to diagnose serosal invasion and nodal metastases of gastric cancer by multidetector-row CT.

BACKGROUND: Multidetector-row CT (MDCT) may provide accurate preoperative staging of resectable gastric cancer. However, the standard methods and criteria to diagnose the T and N stages to select the patients who are good candidates for neoadjuvant chemotherapy have not been established yet. METHODS: The aim of this prospective study was to evaluate the accuracy of MDCT to diagnose the serosal invasion and nodal metastases of gastric cancer. Patients who had gastric adenocarcinoma underwent MDCT scanning using a standardized method. The T and N stage were diagnosed by prespecified criteria. The analyses were performed in the patients who had cN0-2 and M0 tumors and underwent curative gastrectomy as a primary treatment. The accuracy was calculated by comparing the results of MDCT with the histopathological findings. RESULTS: A total of 315 patients were analyzed. The overall diagnostic accuracy (95 % confidence interval) of T staging was 71.4 % (225 of 315, 66.2-76.1). The accuracy, sensitivity, and specificity for serosal invasion were 85.7 % (81.4-89.1), 54.5 % (42.6-66.0), and 94.0 % (90.3-96.3), respectively. The false-positive rate for serosal invasion was 6.0 % (2.9-7.7). The overall diagnostic accuracy of N staging was 75.9 % (239 of 315, 70.9-80.3). The accuracy, sensitivity, and specificity for nodal metastases were 81.3 % (76.6-85.2), 46.4 % (36.8-56.3), and 96.8 % (93.5-98.4), respectively. The false-positive rate for nodal metastases was 3.2 % (1.6-6.5 %). CONCLUSIONS: These results suggest that MDCT provides an accurate diagnosis with high specificity and a low false-positive rate and can be used to select the patients who are candidates for preoperative chemotherapy.

A naked RNA heptamer targeting the human Bcl-2 mRNA induces apoptosis of HL60 leukemia cells.

tRNase Z(L)-utilizing efficacious gene silencing is a gene control technology, which is based on the property that tRNase Z(L) can cleave any target RNA under the direction of an appropriate small guide RNA (sgRNA). To find therapeutic sgRNAs to cure hematological malignancies, we investigated behavior of heptamer-type sgRNA. We demonstrated that a heptamer, mh1(Bcl-2), which targets the human Bcl-2 mRNA, can be taken up by cells without any transfection reagents and that it can induce apoptosis of the leukemia cells. Mouse xenograft experiments showed that a median survival of the mh1(Bcl-2)-treated mice was longer than that of the control mice.

Elimination of specific miRNAs by naked 14-nt sgRNAs.

tRNase Z(L)-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase Z(L), which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5'-half-tRNA, RNA heptamer, hook RNA, and ~14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase Z(L) cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ~50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing.

A potent 2'-O-methylated RNA-based microRNA inhibitor with unique secondary structures.

MicroRNAs (miRNAs) are involved in various biological processes and human diseases. The development of strong low-molecular weight inhibitors of specific miRNAs is thus expected to be useful in providing tools for basic research or in generating promising new therapeutic drugs. We have previously described the development of 'Tough Decoy (TuD) RNA' molecules, which achieve the long-term suppression of specific miRNA activity in mammalian cells when expressed from a lentivirus vector. In our current study, we describe new synthetic miRNA inhibitors, designated as S-TuD (Synthetic TuD), which are composed of two fully 2'-O-methylated RNA strands. Each of these strands includes a miRNA-binding site. Following the hybridization of paired strands, the resultant S-TuD forms a secondary structure with two stems, which resembles the corresponding TuD RNA molecule. By analyzing the effects of S-TuD against miR-21, miR-200c, miR-16 and miR-106b, we have elucidated the critical design features of S-TuD molecules that will provide optimum inhibitory effects following transfection into human cell lines. We further show that the inhibitory effects of a single transfection of S-TuD-miR200c are quite long-lasting (>7 days) and induce partial EMT, the full establishment of which requires 11 days when using a lentivirus vector that expresses TuD-miR200c continuously.

A case of complete resolution of multiple diverticula in the ascending colon through frequent, long distance running in conjunction with the consumption of more vegetables.

Colonic diverticulosis is now a common global disease, but the treatment has conventionally been directed towards its associated complications, such as infection and bleeding, and the treatment of diverticulosis itself has rarely been discussed. This is a case of complete resolution (disappearance) of multiple diverticula in the ascending colon of a Japanese 58-year-old male patient who undertook a frequent, long distance running program for a period of about 8 months. The disappearance was confirmed by barium enema. The patient continued the running program thereafter, and the third barium enema, which was performed 1 year and 3 months after the second, again revealed no diverticula in the ascending colon. He consumed more vegetables after the first barium enema examination (1.52-fold increase). The stools were solid and compact, and effort was needed to void before the running program, but after the running program began, they were loose and easy to void. This case suggests that frequent, vigorous physical exercise, such as long distance running, in conjunction with the consumption of more vegetables can resolve or reduce right-sided colonic diverticula. The decreased intracolonic luminal pressure after running was presumed to be effective.

Identification of SAP155 as the target of GEX1A (Herboxidiene), an antitumor natural product.

GEX1A is a microbial product with antitumor activity. HeLa cells cultured with GEX1A accumulated p27(Kip) and its C-terminally truncated form p27*. GEX1A inhibited the pre-mRNA splicing of p27, producing p27* from the unspliced mRNA containing the first intron. p27* lacked the site required for E3 ligase-mediated proteolysis of p27, leading to its accumulation in GEX1A-treated cells. The accumulated p27* was able to bind to and inhibit the cyclin E-Cdk2 complex that causes E3 ligase-mediated degradation of p27, which probably triggers the accumulation of p27. By using a series of photoaffinity-labeling derivatives of GEX1A, we found that GEX1A targeted SAP155 protein, a subunit of SF3b responsible for pre-mRNA splicing. The linker length between the GEX1A pharmacophore and the photoreactive group was critical for detection of the GEX1A-binding protein. GEX1A serves as a novel splicing inhibitor that specifically impairs the SF3b function by binding to SAP155.

Dynamic susceptibility contrast MRI in advanced pancreatic cancer: semi-automated analysis to predict response to chemotherapy.

The purpose of this study was to assess whether dynamic susceptibility contrast magnetic resonance imaging (DSC-MRI) can predict response to chemotherapy in advanced pancreatic cancer. DSC-MRI was performed using gradient-echo echo-planar imaging after bolus injection of contrast material. Fifty-four patients with advanced pancreatic cancer who were scheduled for chemotherapy were enrolled. ΔR2* was calculated using semi-automated computer analysis capable of tracking moving lesions during DSC-MRI. Pre-treatment maximum ΔR2* and clinical factors including gender, age, tumor stage (UICC III/IV), initial tumor size, and chemotherapy regimen were compared between patients with progressive disease and patients with stable disease as was determined at 3-month follow-up, and between patients with progressive disease and patients with stable disease as was determined at 6-month follow-up. Receiver operating characteristic (ROC) analysis and the Kaplan-Meier method with log-rank test were used to assess the relationship between the pre-treatment maximum ΔR2* and early progression (i.e. at 3-month follow-up). The pre-treatment maximum ΔR2* of patients with disease progression at 3-month follow-up (10.68 ± 3.88 s(-1)) was significantly different (p < 0.01) from that of patients with stable disease at 3-month follow-up (6.94 ± 3.12 s(-1)). Pre-treatment maximum ΔR2* of patients with disease progression at 6-month follow-up was not significantly different from that of patients with stable disease at 6-month follow-up, although a trend was noted (p = 0.08). Pre-treatment clinical factors were not significantly different between progressive and stable patients at 3- and 6-month follow-up. Tumor progression rate was significantly higher in patients with a higher pre-treatment maximum ΔR2* than in those with a lower pre-treatment maximum ΔR2* (median progression time, 38 vs 138 days, p < 0.01, using a cut-off value of 8.13 s(-1) as determined by ROC analysis). In conclusion, DSC-MRI may predict early progression in patients with advanced pancreatic cancer undergoing chemotherapy.

Functional screening identifies a microRNA, miR-491 that induces apoptosis by targeting Bcl-X(L) in colorectal cancer cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate expression of target mRNA. They are involved in many biological processes, including cell proliferation, apoptosis and differentiation, and considered as new therapeutic targets for cancers. In our study, we performed a gain-of-function screen using 319 miRNAs to identify those affecting cell proliferation and death in human colorectal cancer cells (DLD-1). We discovered a number of miRNAs that increased or decreased cell viability in DLD-1. They included known oncogenic miRNAs such as miR-372 and miR-373, and tumor suppressive miRNAs such as miR-124a, but also some for which this information was novel. Among them, miR-491 markedly decreased cell viability by inducing apoptosis. We demonstrated that Bcl-X(L) was a direct target of miR-491, and its silencing contributed to miR-491-induced apoptosis. Moreover, treatment of miR-491 suppressed in vivo tumor growth of DLD-1 in nude mice. Our study provides a new regulation of Bcl-X(L) by miR-491 in colorectal cancer cells, and suggests a therapeutic potential of miRNAs for treating colorectal cancer by targeting Bcl-X(L).

The usefulness of perfusion-weighted magnetic resonance imaging in advanced pancreatic cancer.

OBJECTIVES: Perfusion-weighted magnetic resonance imaging (MRI) can detect the changes of signal intensity in tumors. We evaluated the prognostic value of perfusion-weighted MRI in patients with advanced pancreatic cancer (PC). METHODS: Perfusion-weighted MRI was performed before treatment on 27 consecutive patients with advanced PC. The American Joint Committee on Cancer (AJCC) stages of patients were as follows (8, stage III; 19, stage IV). Imaging acquisition was continually repeated with echo planar sequence every 2 seconds for 2 minutes after a bolus injection of gadolinium. We made a time intensity curve of PC and calculated the signal ratio (SR) on perfusion-weighted imaging. We assessed the relation between SR and clinical factors including tumor stage, lymph node metastasis, liver metastasis, and so on. Patients were divided into low and high SR group and compared SR with the overall survival. RESULTS: All cases showed transient decreases signal intensity (SR, 6.9-55.7%). These patients were classified into 2 groups at cutoff median SR of 22.0% The high SR group significantly correlated with the higher stage (P=0.03) and the presence of lymph node metastasis (P=0.04). The high SR group had significantly shorter overall survival (P=0.04). CONCLUSIONS: Perfusion-weighted MRI may predict the survival in advanced PC patients.

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells.

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of approximately 31 and approximately 60kDa in cell lysates, and the higher expression of approximately 31kDa band was further increased after stimulation with tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS). A approximately 31kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.

Evaluation of dynamic studies of MR mammography for the diagnosis of intraductal lesions with nipple discharge.

OBJECTIVES: We assessed the utility of dynamic magnetic resonance imaging (MRI) in differentiating benign from malignant lesions of the breast and then applied MRI to diagnose intraductal breast tumors with nipple discharge. METHODS: Gadolinium (Gd)-enhanced MR mammography was performed on 74 patients with breast tumors and 8 patients with nipple discharge. RESULTS: The steepest slopes of the contrast medium uptake (S slope) s from time-intensity curves were significantly different between malignant and benign lesions. At S slope threshold of 0.95% /second, malignancy was predicted with a sensitivity and specificity of 75% . Six of 8 cases with nipple discharge were successfully identified by MR ductography by injecting Gd-DTPA into discharging ducts. Among them, 2 non-invasive ductal carcinomas were differentiated from benign lesions by the S slope value. CONCLUSIONS: Dynamic MR mammography is an useful modality for differentiating breast lesions and has potential for evaluating intraductal lesions with nipple discharge.