Low folate status is well recognized as one of the metabolic stressors for colorectal cancer carcinogenesis, but its role in colon cancer invasion remains unknown. Activation of the Sonic hedgehog (Shh) signal in interaction with the transcription nuclear factor-kappa B (NF-κB) pathway is crucial for cancer aggressiveness. The aims of this study were to investigate whether and how folate deprivation promotes invasion by colon cancer cells in relation to Shh signaling and NF-κB pathway activation. Cultivation of epithelial colon carcinoma-derived cells (HCT116) in folate-deficient (FD) medium enhanced cellular migration and invasion, in correlation with epithelial–mesenchymal transition (EMT) associated with Snail expression and E-cadherin suppression, increased production of β1 integrin and increased proteolysis by matrix metalloproteinase 2. Blockade of Shh signaling by cyclopamine (CYC) or of NF-κB activation by BAY abolished FD-enhanced EMT and invasion by HCT116 cells. FD cells had 50–80% less intracellular folate, associated with aberrant hypomethylation of the Shh promoter, than control cells, and increased binding of nuclear NF-κB subunit p65 to the Shh promoter region, which coincided with increased Shh expression and protein production of Shh ligand; in addition, the FD-induced Shh signaling targeted Gli1 transcription activator as well as Ptch receptor. The FD-induced Shh induction and activated signaling were blocked by NF-κB inhibitor BAY. Blockade of Shh signaling abrogated FD-promoted NF-κB activation measured by IκBα degradation and by target gene TNFα expression. Taken together, these findings demonstrate that folate deprivation enhanced invasiveness of colon cancer cells mediated by activation of Shh signaling through promoter hypomethylation and cross actions with the NF-κB pathway.

Previous studies with folate/methyl-deficient rat models proposed the role of folate deficiency in hepatocarcinogenesis and tumour progression. We investigated the relationship between folate status and tumour progression in patients with hepatocellular carcinoma (HCC). Ninety HCC patients (age 62 (sd 10) years) recruited through the Department of Internal Medicine, Chi-Mei Hospital, participated in this cross-sectional study. According to the clinical criteria, 44 % showed marginal folate deficiency (serum folate 7–14 nmol/l; folate intake 278 (sd 212) μg/d), and 16 % were folate deficient ( < 7 nmol/l; 207 (sd 113) μg/d). Serum folate showed inverse correlations with three elements of tumour progression: tumour size (r − 0·29; P = 0·005), tumour multiplicity (r − 0·24; P = 0·018) and metastasis (r − 0·39; P = 0·0001). When HCC progression was categorised into stages I to IV, serum folate decreased as HCC stage progressed (stage I, 24·5 (sd 11·5); stage IV, 10·3 (sd 3·3) nmol/l; P = 0·032). After adjustment for age, sex, lifestyle and dietary factors, patients with low blood folate status (serum folate < 14 nmol/l) had increased risks for advanced tumour progression in large tumours (OR 7·1 (95 % CI 2·27, 21·9); P = 0·0007), tumour multiplicity (OR 3·2 (95 % CI 1·07, 3·51); P = 0·004) and metastasis (OR 4·5 (95 % CI 1·11, 18·4); P = 0·03) relative to those with normal folate status. Further controlling for liver injury, tumour proliferation and tumour stage, however, negated the effect of folate on advanced tumour progression. The data thus suggest that low blood folate status could be a risk factor for tumour progression, which is modulated by clinical lesions present in HCC patients. Future studies with larger sample sizes are warranted to explore the joint effects of low folate and hepatic lesions in human HCC malignancy.

The mechanistic role of colonic low folate metabolic stress (LFMS) in colorectal cancer (CRC) malignancy development remains unknown. Folate analysis on the 99 paired human CRC tissues localized LFMS to the deep invasive T3/T4 staged tumours with hypo‐methylated sonic hedgehog (Shh) promoter region and amplified expressions of Shh ligand and Gli1 effector, which coincided with deregulated expressions of the epithelial‐mesenchymal transition (EMT) mediators. Colonic folate levels of CRC were inversely correlated with pluripotent expressions of the SOX2, NANOG and OCT4 markers (p < 0.05). Exposure of human colon adenocarcinoma cells to LFMS microenvironment significantly hypomethylated Shh promoter region, activated Shh signaling, induced transcript and protein expressions of the pluripotent markers, promoted trans‐differentiation as EMT by deregulation of Snail mediator and epithelial marker E‐cadherin, increased MMP2/MMP9 enzymatic digestion on matrix protein for invasion, and promoted self‐renewal capability of anchorage‐independent tumor‐spheroid formation. LFMS‐induced cancer stem cell (CSC) signature and CRC invasion is synergized with inhibition of DNA methylation by 5‐Aza‐2‐deoxycytidine (5AZA) in rewiring EMT genotypes, which can be blockade by the Shh inhibitor (cyclopamine). The in vivo and in vitro data corroboratively identify CSC‐like molecular targets specific to the LFMS‐predisposed invasive CRC through reprogramming DNA methylation‐activated Shh signaling. The study highlights CSC targets specific to LFMS‐predisposed invasive CRC in optimizing folate co‐chemotherapy to minimize tumour metastasis potential of CRC patients.

Mitochondrial (mt) DNA deletions and low folate status, proposed characteristics of carcinogenesis, in relation to human hepatocellular carcinoma (HCC) susceptibility are not clearly understood. We hypothesised that low folate status may modify frequencies of mtDNA deletions in humans, both of which could predispose individuals to HCC development. Biochemical folate status of serum and lymphocytes, and frequencies of mtDNA deletions in lymphocytes were determined in ninety HCC cases and ninety cancer-free healthy controls, individually matched by age and sex. The data revealed that HCC patients had lower levels of serum folate (P = 0·0002), lymphocytic folate (P = 0·040) and accumulated higher frequency of lymphocytic mtDNA deletions (P < 0·0001) than the controls. In the total studied subjects, frequencies of lymphocytic mtDNA deletions were associated with hepatitic B infection (P = 0·004) and HCC incidents (P = 0·001), and were correlated with serum folate (r− 0·155;P = 0·041), lymphocyte folate (r− 0·314;P = 0·0001), levels of glutamate-oxaloacetate transaminase (GOT) (r0·206;P = 0·006), glutamate-pyruvate transaminase (GPT) (r0·163;P = 0·037) and α-fetal protein concentrations (r0·212;P = 0·005). After adjustment for age, sex, lifestyle and one-carbon metabolite factors, individuals with low blood folate ( < 11·5 nmol/l) or high mtDNA deletions (Δ threshold cycle number (C_t)>5·3) had increased risks for HCC (OR 7·7, 95 % CI 1·9, 29·9,P = 0·003; OR 5·4; 95 % CI 1·7, 16·8,P = 0·003, respectively). When combined with folate deficiency (serum folate < 14 nmol/l), the OR of HCC in individuals with high levels of lymphocytic mtDNA deletions was enhanced (OR 13·3; 95 % CI 1·45, 122;P = 0·008). Further controlling for GOT and GPT levels, however, negated those effects on HCC risk. Taken together, the data suggest that biochemical folate status and liver injuries are important modulators to lymphocytic mtDNA deletions. The mt genetic instability that results from a high rate of mtDNA deletions and/or low folate status increased the risk for HCC, which is mediated by clinical hepatic lesions.