Effects of High-Mobility Group Box-1 on Mucosal Immunity and Epithelial Differentiation in Colitic Carcinoma.

Abnormalities in mucosal immunity are involved in the onset and progression of ulcerative colitis (UC), resulting in a high incidence of colorectal cancer (CRC). While high-mobility group box-1 (HMGB1) is overexpressed during colorectal carcinogenesis, its role in UC-related carcinogenesis remains unclear. In the present study, we investigated the role of HMGB1 in UC-related carcinogenesis and sporadic CRC. Both the azoxymethane colon carcinogenesis and dextran sulfate sodium colitis carcinogenesis models demonstrated temporal increases in mucosal HMGB1 levels. Activated CD8+ cells initially increased and then decreased, whereas exhausted CD8+ cells increased. Additionally, we observed increased regulatory CD8+ cells, decreased naïve CD8+ cells, and decreased mucosal epithelial differentiation. In the in vitro study, HMGB1 induced energy reprogramming from oxidative phosphorylation to glycolysis in CD8+ cells and intestinal epithelial cells. Furthermore, in UC dysplasia, UC-related CRC, and hyperplastic mucosa surrounding human sporadic CRC, we found increased mucosal HMGB1, decreased activated CD8+ cells, and suppressed mucosal epithelial differentiation. However, we observed increased activated CD8+ cells in active UC mucosa. These findings indicate that HMGB1 plays an important role in modulating mucosal immunity and epithelial dedifferentiation in both UC-related carcinogenesis and sporadic CRC.

Cancerous Conditions Accelerate the Aging of Skeletal Muscle via Mitochondrial DNA Damage.

Skeletal muscle aging and sarcopenia result in similar changes in the levels of aging markers. However, few studies have examined cancer sarcopenia from the perspective of aging. Therefore, this study investigated aging in cancer sarcopenia and explored its causes in vitro and in vivo. In mouse aging, in vitro cachexia, and mouse cachexia models, skeletal muscles showed similar changes in aging markers including oxidative stress, fibrosis, reduced muscle differentiation potential, and telomere shortening. Furthermore, examination of mitochondrial DNA from skeletal muscle revealed a 5 kb deletion in the major arc; truncation of complexes I, IV, and V in the electron transport chain; and reduced oxidative phosphorylation (OXPHOS). The mouse cachexia model demonstrated high levels of high-mobility group box-1 (HMGB1) and tumor necrosis factor-α (TNFα) in cancer ascites. Continuous administration of neutralizing antibodies against HMGB1 and TNFα in this model reduced oxidative stress and abrogated mitochondrial DNA deletion. These results suggest that in cancer sarcopenia, mitochondrial oxidative stress caused by inflammatory cytokines leads to mitochondrial DNA damage, which in turn leads to decreased OXPHOS and the promotion of aging.

Berberine Improves Cancer-Derived Myocardial Impairment in Experimental Cachexia Models by Targeting High-Mobility Group Box-1.

Cardiac disorders in cancer patients pose significant challenges to disease prognosis. While it has been established that these disorders are linked to cancer cells, the precise underlying mechanisms remain elusive. In this study, we investigated the impact of cancerous ascites from the rat colonic carcinoma cell line RCN9 on H9c2 cardiomyoblast cells. We found that the ascites reduced mitochondrial volume, increased oxidative stress, and decreased membrane potential in the cardiomyoblast cells, leading to apoptosis and autophagy. Although the ascites fluid contained a substantial amount of high-mobility group box-1 (HMGB1), we observed that neutralizing HMGB1 with a specific antibody mitigated the damage inflicted on myocardial cells. Our mechanistic investigations revealed that HMGB1 activated both nuclear factor κB and phosphoinositide 3-kinases-AKT signals through HMGB1 receptors, namely the receptor for advanced glycation end products and toll-like receptor-4, thereby promoting apoptosis and autophagy. In contrast, treatment with berberine (BBR) induced the expression of miR-181c-5p and miR-340-5p while suppressing HMGB1 expression in RCN9 cells. Furthermore, BBR reduced HMGB1 receptor expression in cardiomyocytes, consequently mitigating HMGB1-induced damage. We validated the myocardial protective effects of BBR in a cachectic rat model. These findings underscore the strong association between HMGB1 and cancer cachexia, highlighting BBR as a promising therapeutic agent for myocardial protection through HMGB1 suppression and modulation of the signaling system.

Nuclear MAST4 Suppresses FOXO3 through Interaction with AKT3 and Induces Chemoresistance in Pancreatic Ductal Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line-MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.

Involvement of Ferroptosis Induction and Oxidative Phosphorylation Inhibition in the Anticancer-Drug-Induced Myocardial Injury: Ameliorative Role of Pterostilbene.

Patients with cancer die from cardiac dysfunction second only to the disease itself. Cardiotoxicity caused by anticancer drugs has been emphasized as a possible cause; however, the details remain unclear. To investigate this mechanism, we treated rat cardiomyoblast H9c2 cells with sunitinib, lapatinib, 5-fluorouracil, and cisplatin to examine their effects. All anticancer drugs increased ROS, lipid peroxide, and iron (II) levels in the mitochondria and decreased glutathione peroxidase-4 levels and the GSH/GSSG ratio. Against this background, mitochondrial iron (II) accumulates through the unregulated expression of haem oxygenase-1 and ferrochelatase. Anticancer-drug-induced cell death was suppressed by N-acetylcysteine, deferoxamine, and ferrostatin, indicating ferroptosis. Anticancer drug treatment impairs mitochondrial DNA and inhibits oxidative phosphorylation in H9c2 cells. Similar results were observed in the hearts of cancer-free rats treated with anticancer drugs in vitro. In contrast, treatment with pterostilbene inhibited the induction of ferroptosis and rescued the energy restriction induced by anticancer drugs both in vitro and in vivo. These findings suggest that induction of ferroptosis and inhibition of oxidative phosphorylation are mechanisms by which anticancer drugs cause myocardial damage. As pterostilbene ameliorates these mechanisms, it is expected to have significant clinical applications.

Digital PCR using a simple PDMS microfluidic chip and standard laboratory equipment.

Digital PCR (dPCR) enables sensitive and precise quantification of template nucleic acid without calibration. However, dPCR is not yet in widespread use, probably due to the need for expensive specialized instruments. In this paper, we describe a dPCR system using a simple microfluidic chip and common laboratory tools. The microfluidic chip consists of two parts: a PDMS part with 24,840 × 0.25 nL microwells and a PDMS-coated flat glass plate. Human RNase P gene was adopted as the model template. Commercial products of human genomic DNA and real-time PCR reagents were mixed to make a PCR mixture. The PCR mixture was confined to the microwells by the PDMS degas-driven liquid control technique. The thermal cycling was performed on a common well-type thermal cycler with a minor modification. During the thermal cycling, evaporation of the PCR mixture was prevented with a handmade water holder. In the fluorescence image, bright (positive) microwells and dim (negative) ones were clearly discriminated. The number of the positive microwells was counted using software, and was used for estimation of the template concentration in the sample based on the theory of the Poisson distribution. The estimated concentrations well agreed with the input template concentrations in the range from 1.32 copies/µL to 13 200 copies/µL. The techniques presented in this paper will pave the way for facile dPCR in a broad range of laboratories without the need for expensive instruments.

Role of creatine shuttle in colorectal cancer cells.

The creatine shuttle translocates the energy generated by oxidative phosphorylation to the cytoplasm via mitochondrial creatine kinase (MTCK) and creatine kinase B (CKB) in the cytoplasm. It is not apparent how the creatine shuttle is related to cancer. Here, we analyzed the expression and function of CKB and MTCK in colorectal cancer (CRC) and investigated the role of the creatine shuttle in CRC. Compared with normal mucosa, 184 CRC tissues had higher levels of CKB and MTCK, and these levels were associated with histological grade, tumor invasion, and distant metastasis. CK inhibitor dinitrofluorobenzene (DNFB) on CRC cell lines HT29 and CT26 inhibited cell proliferation and stemness to less than 2/3 and 1/20 of their control levels, respectively. In this treatment, the production of reactive oxygen species increased, mitochondrial respiration decreased, and mitochondrial volume and membrane potential decreased. In a syngeneic BALB/c mouse model using CT26 cells pretreated with DNFB, peritoneal metastasis was suppressed to 70%. Phosphorylation of EGFR, AKT, and ERK1/2 was inhibited in DNFB-treated tumors. High ATP concentrations prevented EGFR phosphorylation in HT29 cells following DNFB treatment, CKB or MTCK knockdown, and cyclocreatine administration. Despite not being immunoprecipitated, CKB and EGFR were brought closer together by EGF stimulation. These findings imply that blocking the creatine shuttle decreases the energy supply, suppresses oxidative phosphorylation, and blocks ATP delivery to phosphorylation signals, preventing signal transduction. These findings highlight the critical role of the creatine shuttle in cancer cells and suggest a potential new cancer treatment target.

Lauric Acid Overcomes Hypoxia-Induced Gemcitabine Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Although gemcitabine (GEM) is widely used in chemotherapy for pancreatic ductal adenocarcinoma (PDA), drug resistance restricts its clinical effectiveness. To examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced energy production and decreased mitochondrial reactive oxygen species levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. Inhibition of hypoxia-inducible factor-1α in both cell lines did not restore the GEM sensitivity. In contrast, treatment of both cell types with lauric acid (LAA), a medium-chain fatty acid, restored GEM sensitivity. These results suggest that decreased energy production, decreased mitochondrial reactive oxygen species levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of oxidative phosphorylation by LAA could be a tool to overcome GEM resistance. Clinical verification of the effectiveness of LAA in GEM resistance is necessary in the future.

Short Telomere Lesions with Dysplastic Metaplasia Histology May Represent Precancerous Lesions of Helicobacter pylori-Positive Gastric Mucosa.

Gastric cancers are strongly associated with Helicobacter pylori infection, with intestinal metaplasia characterizing the background mucosa in most cases. However, only a subset of intestinal metaplasia cases proceed to carcinogenesis, and the characteristics of high-risk intestinal metaplasia that link it with gastric cancer are still unclear. We examined telomere reduction in five gastrectomy specimens using fluorescence in situ hybridization, and identified areas with localized telomere loss (outside of cancerous lesions), which were designated as short telomere lesions (STLs). Histological analyses indicated that STLs were characteristic of intestinal metaplasia accompanied by nuclear enlargement but lacking structural atypia, which we termed dysplastic metaplasia (DM). A review of gastric biopsy specimens from 587 H. pylori-positive patients revealed 32 cases of DM, 13 of which were classified as high-grade based on the degree of nuclear enlargement. All high-grade DM cases exhibited a telomere volume reduced to less than 60% of that of lymphocytes, increased stemness, and telomerase reverse transcriptase (TERT) expression. Two patients (15%) exhibited low levels of p53 nuclear retention. After a 10-year follow-up, 7 (54%) of the high-grade DM cases had progressed to gastric cancer. These results suggest that DM is characterized by telomere shortening, TERT expression, and stem cell proliferation, and high-grade DM is a high-grade intestinal metaplasia that likely represents a precancerous lesion of gastric cancer. High-grade DM is expected to effectively prevent progression to gastric cancer in H. pylori-positive patients.

An Axis between the Long Non-Coding RNA HOXA11-AS and NQOs Enhances Metastatic Ability in Oral Squamous Cell Carcinoma.

Long non-coding RNAs (lncRNAs) play critical roles in human cancers. HOXA11 anti-sense RNA (HOXA11-AS) is an lncRNA belonging to the homeobox (HOX) gene cluster that promotes liver metastasis in human colon cancer. However, its role and mechanism of action in human oral squamous cell carcinoma (OSCC) are unclear. In this study, we investigated HOXA11-AS expression and function in human OSCC tissues and cell lines, as well as a mouse model of OSCC. Our analyses showed that HOXA11-AS expression in human OSCC cases correlates with lymph node metastasis, nicotinamide adenine dinucleotide (NAD)(P)H: quinone oxidoreductase 1 (NQO1) upregulation, and dihydronicotinamide riboside (NRH): quinone oxidoreductase 2 (NQO2) downregulation. Using the human OSCC cell lines HSC3 and HSC4, we demonstrate that HOXA11-AS promotes NQO1 expression by sponging microRNA-494. In contrast, HOXA11-AS recruits zeste homolog 2 (EZH2) to the NQO2 promoter to suppress its expression via the trimethylation of H3K27. The upregulation of NQO1 enzymatic activity by HOXA11-AS results in the consumption of flavin adenine dinucleotide (FAD), which reduces FAD-requiring glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity and suppresses glycolysis. However, our analyses show that lactic acid fermentation levels are preserved by glutaminolysis due to increased malic enzyme-1 expression, promoting enhanced proliferation, invasion, survival, and drug resistance. In contrast, suppression of NQO2 expression reduces the consumption of NRH via NQO2 enzymatic activity and increases NAD levels, which promotes enhanced stemness and metastatic potential. In mouse tumor models, knockdown of HOXA11-AS markedly suppressed tumor growth and lung metastasis. From these findings, targeting HOXA11-AS may strongly suppress high-grade OSCC by regulating both NQO1 and NQO2.

Gemcitabine Resistance in Pancreatic Ductal Carcinoma Cell Lines Stems from Reprogramming of Energy Metabolism.

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis because it is often detected at an advanced stage, and drug resistance interferes with treatment. However, the mechanism underlying drug resistance in PDAC remains unclear. Here, we investigated metabolic changes between a parental PDAC cell line and a gemcitabine (GEM)-resistant PDAC cell line. We established a GEM-resistant cell line, MIA-G, from MIA-PaCa-2 parental (MIA-P) cells using continuous therapeutic-dose GEM treatment. MIA-G cells were also more resistant to 5-fluorouracil in comparison to MIA-P cells. Metabolic flux analysis showed a higher oxygen consumption rate (OCR) in MIA-G cells than in MIA-P cells. Notably, OCR was suppressed by GEM treatment only in MIA-G cells. GEM treatment increased mitochondrial membrane potential and mitochondrial reactive oxygen species (ROS) in MIA-P cells, but not in MIA-G cells. Glutamine uptake and peroxidase levels were elevated in MIA-G cells. The antioxidants N-acetyl-L-cysteine and vitamin C increased the sensitivity to GEM in both cell lines. In MIA-G cells, the expression of the mitochondrial transcription factor A also decreased. Furthermore, rotenone reduced the sensitivity of MIA-P cells to GEM. These findings suggest that the suppression of oxidative phosphorylation contributes to GEM resistance by reducing ROS production. Our study provides a new approach for reducing GEM resistance in PDAC.

Hypomethylation of CLDN4 Gene Promoter Is Associated with Malignant Phenotype in Urinary Bladder Cancer.

The tight junction (TJ) protein claudin-4 (CLDN4) is overexpressed in bladder urothelial carcinoma (BUC) and correlates with cancer progression. However, the mechanism of CLDN4 upregulation and promotion of malignant phenotype is not clear. Here, we analyzed 157 cases of BUC and investigated the hypomethylation of CpG island in the CLDN4 promoter DNA and its correlation with cancer progression. In hypomethylated cases, CLDN4 expression, cell proliferation, stemness, and epithelial-mesenchymal transition were increased. Treatment of three human BUC cell lines with the demethylating agent aza-2'-deoxycytidine (AZA) led to excessive CLDN4 expression, and, specifically, to an increase in CLDN4 monomer that is not integrated into the TJ. The TJ-unintegrated CLDN4 was found to bind integrin ß1 and increase stemness, drug resistance, and metastatic ability of the cells as well as show an anti-apoptosis effect likely via FAK phosphorylation, which reduces upon knockdown of CLDN4. Thus, CLDN4 is overexpressed in BUC by an epigenetic mechanism and the high expression enhances the malignant phenotype of BUC via increased levels of TJ-unintegrated CLDN4. CLDN4 promoter DNA methylation is expected to be a novel indicator of BUC malignant phenotype and a new therapeutic target.

Oxidized high mobility group B-1 enhances metastability of colorectal cancer via modification of mesenchymal stem/stromal cells.

High mobility group box-1 (HMGB1) is known to be a chemotactic factor for mesenchymal stem/stromal cells (MSCs), but the effect of post-translational modification on its function is not clear. In this study, we hypothesized that differences in the oxidation state of HMGB1 would lead to differences in the function of MSCs in cancer. In human colorectal cancer, MSCs infiltrating into the stroma were correlated with liver metastasis and serum HMGB1. In animal models, oxidized HMGB1 mobilized three-fold fewer MSCs to subcutaneous tumors compared with reduced HMGB1. Reduced HMGB1 inhibited the proliferation of mouse bone marrow MSCs (BM-MSCs) and induced differentiation into osteoblasts and vascular pericytes, whereas oxidized HMGB1 promoted proliferation and increased stemness, and no differentiation was observed. When BM-MSCs pretreated with oxidized HMGB1 were co-cultured with syngeneic cancer cells, cell proliferation and stemness of cancer cells were increased, and tumorigenesis and drug resistance were promoted. In contrast, co-culture with reduced HMGB1-pretreated BM-MSCs did not enhance stemness. In an animal orthotopic transplantation colorectal cancer model, oxidized HMGB1, but not reduced HMGB1, promoted liver metastasis with intratumoral MSC chemotaxis. Therefore, oxidized HMGB1 reprograms MSCs and promotes cancer malignancy. The oxidized HMGB1-MSC axis may be an important target for cancer therapy.

Suppressive GLI2 fragment enhances liver metastasis in colorectal cancer.

Linoleic acid (LA) has been shown to cause inflammation and promote development of colorectal cancer (CRC). Moreover, many literatures show that LA is associated with cancer metastasis. Metastatic cancer cells have high stemness, suggesting that LA might affect the stemness of cancer cells. In this study, we examined the effect of LA on the hedgehog system, which affects cancer stemness. In CT26 cells, LA treatment induced the expression of sonic hedgehog (Shh); the signal transduction factor, and glioma-associated oncogene homolog (Gli) 2, whereas the expression of SRY-box transcription factor (Sox) 17 was suppressed. Furthermore, LA reduced GLI2 ubiquitination, resulting in an increase in the N-terminal fragment of GLI2, known as suppressive GLI2, produced by cleavage of GLI2. LA-induced cleaved GLI2 was also detected in Colo320 and HT29 human CRC cells. Knocking down Gli2 abrogated the LA-mediated suppression of Sox17 expression. These results suggest that LA promotes tumor cell stemness by increasing of suppressive GLI2 fragments via GLI2 modification. In mouse liver metastasis models, LA enhanced metastasis with production of the suppressive GLI2 fragments in CT26 and HT29 cells, whereas knockdown of GLI2 abrogated LA-induced metastatic activity. In human CRCs, the cases with liver metastasis showed the suppressive GLI2 fragments. This study provides mechanistic insights into LA-induced stemness in colon cancer cells. This finding suggests that dietary intake of LA might increase the stemness of cancer cells and enhance metastatic activity of the cancer.

BRAF Mutation Is Associated with Hyperplastic Polyp-Associated Gastric Cancer.

Gastric hyperplastic polyps (GHP) are frequently found to be benign polyps and have been considered to have a low carcinogenic potential. The characteristics of the hyperplastic polyp-associated gastric cancer (HPAGC) remain unclear. Therefore, we analyzed samples from 102 GHP patients and identified 20 low-grade atypical GHPs (19.6%), 7 high-grade atypical GHPs (6.9%), and 5 intramucosal cancer samples (4.9%). GHP atypia was more common in the elderly and increased with increasing polyp size. In particular, polyps larger than 1 cm were associated with a higher grade and cancer. Furthermore, mucus production decreased with increasing atypia. Although no correlation was found between atypia and Helicobacter pylori infection or intestinal metaplasia, enhanced proliferative ability (Ki-67) did correlate with atypia, as did nuclear 8-hydroxy-2'-deoxyguanosine levels. Interestingly, 4-hydroxynonenal levels in granulation tissue and the area ratio of granulation tissue within polyps also correlated with GHP atypia. In five cases of HPAGC, three cases exhibited caudal type homeobox transcription factor (CDX2)-positive cells and a mixed mucin phenotype, which is considered to be related to H. pylori infection. By contrast, two cases were CDX2 negative, with a gastric mucin phenotype, and H. pylori infection was not observed in the tumor or the surrounding mucosa. In these cases, a v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation (V600E) was detected. All cancer samples showed high stemness and p53 protein accumulation, but no KRAS mutations. The molecular and phenotypic characteristics of the cases characterized by BRAF mutations may represent a novel subtype of HPAGC, reflecting a conserved pathway to oncogenesis that does not involve H. pylori infection. These findings are worthy of further investigation in a large-scale study with a substantial cohort of HPAGC patients to establish their clinical significance.

Enhancement of Anti-Tumoral Immunity by ß-Casomorphin-7 Inhibits Cancer Development and Metastasis of Colorectal Cancer.

ß-Casomorphin-7 (BCM) is a degradation product of ß-casein, a milk component, and has been suggested to affect the immune system. However, its effect on mucosal immunity, especially anti-tumor immunity, in cancer-bearing individuals is not clear. We investigated the effects of BCM on lymphocytes using an in vitro system comprising mouse splenocytes, a mouse colorectal carcinogenesis model, and a mouse orthotopic colorectal cancer model. Treatment of mouse splenocytes with BCM in vitro reduced numbers of cluster of differentiation (CD) 20+ B cells, CD4+ T cells, and regulatory T cells (Tregs), and increased CD8+ T cells. Administration of BCM and the CD10 inhibitor thiorphan (TOP) to mice resulted in similar alterations in the lymphocyte subsets in the spleen and intestinal mucosa. BCM was degraded in a concentration- and time-dependent manner by the neutral endopeptidase CD10, and the formed BCM degradation product did not affect the lymphocyte counts. Furthermore, degradation was completely suppressed by TOP. In the azoxymethane mouse colorectal carcinogenesis model, the incidence of aberrant crypt foci, adenoma, and adenocarcinoma was reduced by co-treatment with BCM and TOP. Furthermore, when CT26 mouse colon cancer cells were inoculated into the cecum of syngeneic BALB/c mice and concurrently treated with BCM and TOP, infiltration of CD8+ T cells was promoted, and tumor growth and liver metastasis were suppressed. These results suggest that by suppressing the BCM degradation system, the anti-tumor effect of BCM is enhanced and it can suppress the development and progression of colorectal cancer.

Linoleic Acid Upregulates Microrna-494 to Induce Quiescence in Colorectal Cancer.

Cancer dormancy is a state characterized by the quiescence of disseminated cancer cells, and tumor recurrence occurs when such cells re-proliferate after a long incubation period. These cancer cells tend to be treatment resistant and one of the barriers to successful therapeutic intervention. We have previously reported that long-term treatment of cancer cells with linoleic acid (LA) induces a dormancy-like phenotype. However, the mechanism underpinning this effect has not yet been clarified. Here, we investigate the mechanism of LA-induced quiescence in cancer cells. We first confirmed that long-term treatment of the mouse colorectal cancer cell line CT26 with LA induced quiescence. When these cells were inoculated subcutaneously into a syngeneic mouse and fed with an LA diet, the inoculated cancer cells maintained the quiescent state and exhibited markers of dormancy. LA-treated CT26 cells showed reduced oxidative phosphorylation, glycolysis, and energy production as well as reduced expression of the regulatory factors Pgc1α and MycC. MicroRNA expression profiling revealed that LA induced an upregulation in miR-494. The expression of Pgc1α and MycC were both induced by an miR-494 mimic, and the LA-induced decrease in gene expression was abrogated by an miR-494 inhibitor. The expression of miR-494 was enhanced by the mitochondrial oxidative stress produced by LA. In a syngeneic mouse subcutaneous tumor model, growth suppression by an LA diet and growth delay by LA pretreatment + LA diet were found to have similar effects as administration of an miR-494 mimic. In contrast, the effects of LA were abrogated by an miR-494 inhibitor. Analysis of human colorectal cancer tissue revealed that miR-494 was present at low levels in non-metastatic cases and cases with simultaneous liver metastases but was expressed at high levels in cases with delayed liver metastases, which also exhibited reduced expression of PGC1α and MYCC. These results suggest that miR-494 is involved in cancer dormancy induced by high levels of LA intake and that this microRNA may be valuable in targeting dormant cancer cells.

Malic Enzyme 1 Is Associated with Tumor Budding in Oral Squamous Cell Carcinomas.

Budding at the tumor invasive front has been correlated with the malignant properties of many cancers. Malic enzyme 1 (ME1) promotes the Warburg effect in cancer cells and induces epithelial-mesenchymal transition (EMT) in oral squamous cell carcinoma (OSCC). Therefore, we investigated the role of ME1 in tumor budding in OSCC. Tumor budding was measured in 96 human OSCCs by immunostaining for an epithelial marker (AE1/AE3), and its expression was compared with that of ME1. A significant correlation was observed between tumor budding and ME1 expression. The correlation increased with the progression of cancer. In human OSCC cells, lactate secretion decreased when lactate fermentation was suppressed by knockdown of ME1 and lactate dehydrogenase A or inhibition of pyruvate dehydrogenase (PDH) kinase. Furthermore, the extracellular pH increased, and the EMT phenotype was suppressed. In contrast, when oxidative phosphorylation was suppressed by PDH knockdown, lactate secretion increased, extracellular pH decreased, and the EMT phenotype was promoted. Induction of chemical hypoxia in OSCC cells by CoCl2 treatment resulted in increased ME1 expression along with HIF1α expression and promotion of the EMT phenotype. Hypoxic conditions also increased matrix metalloproteinases expression and decreased mitochondrial membrane potential, mitochondrial oxidative stress, and extracellular pH. Furthermore, the hypoxic treatment resulted in the activation of Yes-associated protein (YAP), which was abolished by ME1 knockdown. These findings suggest that cancer cells at the tumor front in hypoxic environments increase their lactate secretion by switching their energy metabolism from oxidative phosphorylation to glycolysis owing to ME1 overexpression, decrease in extracellular pH, and YAP activation. These alterations enhance EMT and the subsequent tumor budding. Tumor budding and ME1 expression are thus considered useful markers of OSCC malignancy, and ME1 is expected to be a relevant target for molecular therapy.

Evaluation of cancer-derived myocardial impairments using a mouse model.

Myocardial damage in cancer patients is emphasized as a cause of death; however, there are not many murine cachexia models to evaluate cancer-derived heart disorder. Using the mouse cachexia model that we established previously, we investigated myocardial damage in tumor-bearing mice. In cachexic mice, decreased heart weight and myocardial volume, and dilated left ventricular lumen, and atrophied cardiomyocytes were noted. The cardiomyocytes also showed accumulated 8-hydroxydeoxyguanosine, decreased leucine zipper and EF-hand-containing transmembrane protein-1, and increased microtubule-associated protein light chain3-II. Levels of tumor necrosis factor-α and high-mobility group box-1 proteins in the myocardium were increased, and nuclear factor κB, a signaling molecule associated with these proteins, was activated. When rat cardiomyoblasts (H9c2 cells) were treated with mouse cachexia model ascites and subjected to flux analysis, both oxidative phosphorylation and glycolysis were suppressed, and the cells were in a quiescent state. These results are in good agreement with those previously reported on cancerous myocardial damage. The established mouse cachexia model can therefore be considered useful for analyzing cancer-derived myocardial damage.