guanosine-triphosphate and Colorectal-Neoplasms

Emerging evidence shows that KRAS-mutant colorectal cancer (CRC) depends on glutamine (Gln) for survival and progression, indicating that targeting Gln metabolism may be a promising therapeutic strategy for KRAS-mutant CRC. However, the precise mechanism by which Gln metabolism reprogramming promotes and coordinates KRAS-mutant CRC progression remains to be fully investigated. Here, we discovered that solute carrier 25 member 21 (SLC25A21) expression was downregulated in KRAS-mutant CRC, and that SLC25A21 downregulation was correlated with poor survival of KRAS-mutant CRC patients. SLC25A21 depletion selectively accelerated the growth, invasion, migration, and metastasis of KRAS-mutant CRC cells in vitro and in vivo, and inhibited Gln-derived α-ketoglutarate (α-KG) efflux from mitochondria, thereby potentiating Gln replenishment, accompanied by increased GTP availability for persistent KRAS activation in KRAS-mutant CRC. The restoration of SLC25A21 expression impaired the KRAS-mutation-mediated resistance to cetuximab in KRAS-mutant CRC. Moreover, the arrested α-KG efflux that occurred in response to SLC25A21 depletion inhibited the activity of α-KG-dependent DNA demethylases, resulting in a further decrease in SLC25A21 expression. Our studies demonstrate that SLC25A21 plays a significant role as a tumor suppressor in KRAS-mutant CRC by antagonizing Gln-dependent anaplerosis to limit GTP availability for KRAS activation, which suggests potential alternative therapeutic strategies for KRAS-mutant CRC.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Down-Regulation; Glutamine; Guanosine Triphosphate; Humans; Proto-Oncogene Proteins p21(ras)

Topics: Amino Acid Substitution; Antineoplastic Agents; Catalytic Domain; Cell Line; Colorectal Neoplasms; ErbB Receptors; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Models, Molecular; Neurofibromin 1; Protein Kinase Inhibitors; Proto-Oncogene Proteins p21(ras)

The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase-regulated nuclear-cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage-induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β-dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP-regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage.

Topics: beta Karyopherins; Cell Cycle; Cell Cycle Proteins; Cellular Senescence; Colorectal Neoplasms; DNA Damage; DNA Repair; Doxorubicin; Drug Resistance, Neoplasm; Exportin 1 Protein; Gene Expression Regulation; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; HCT116 Cells; HeLa Cells; Humans; Karyopherins; Nuclear Proteins; ran GTP-Binding Protein; Receptors, Cytoplasmic and Nuclear; RNA Interference

RNA editing can increase RNA sequence variation without altering the DNA sequence. By comparing whole-genome and transcriptome sequence data of a rectal cancer, we found novel tumor-associated increase of RNA editing in ras homologue family member Q (RHOQ) transcripts. The adenosine-to-inosine (A-to-I) editing results in substitution of asparagine with serine at residue 136. We observed a higher level of the RHOQ RNA editing in tumor compared with normal tissue in colorectal cancer (CRC). The degree of RNA editing was associated with RhoQ protein activity in CRC cancer cell lines. RhoQ N136S amino acid substitution increased RhoQ activity, actin cytoskeletal reorganization, and invasion potential. KRAS mutation further increased the invasion potential of RhoQ N136S in vitro. Among CRC patients, recurrence was more frequently observed in patients with tumors having edited RHOQ transcripts and mutations in the KRAS gene. In summary, we show that RNA editing is another mechanism of sequence alteration that contributes to CRC progression.

Topics: Actins; Amino Acid Sequence; Animals; Base Sequence; Cell Line, Tumor; Chlorocebus aethiops; Colorectal Neoplasms; COS Cells; Guanosine Triphosphate; Humans; Male; Middle Aged; Models, Molecular; Molecular Sequence Data; Neoplasm Invasiveness; rho GTP-Binding Proteins; RNA Editing; RNA, Messenger

Colorectal cancer cells exhibit limited cytotoxicity towards Tiazofurin, a pro-drug metabolized by cytosolic nicotinamide mononucleotide adenylyltransferase2 (NMNAT2) to thiazole-4-carboxamide adenine dinucleotide, a potent inhibitor of inosine 5'-monophosphate dehydrogenase required for cellular guanylate synthesis. We tested the hypothesis that colorectal cancer cells that exhibit low levels of NMNAT2 and are refractory to Tiazofurin can be rendered sensitive to Tiazofurin by overexpressing NMNAT2. Transfection of hNMNAT2 resulted in a six- and threefold cytoplasmic overexpression in Caco2 and HT29 cell lines correlating with Tiazofurin-induced enhanced cell-kill. Folate receptors expressed on the cell surface of 30-50% colorectal carcinomas were exploited for cellular targeting with Tiazofurin encapsulated in folate-tethered nanoparticles. Our results indicated that in wild-type colorectal cancer cells, free Tiazofurin-induced EC50 cell-kill was 1500-2000 μM, which was reduced to 66-156 μM in hNMNAT2-overexpressed cells treated with Tiazofurin encapsulated in non-targeted nanoparticles. This efficacy was improved threefold by encapsulating Tiazofurin in folate-tethered nanoparticles to obtain an EC(50) cell-kill of 22-59 μM, an equivalent of 100-300 mg m(-2) (one-tenth of the approved dose of Tiazofurin in humans), which will result in minimal toxicity leading to cancer cell-kill. This proof-of-principle study suggests that resistance of colorectal cancer cell-kill to Tiazofurin can be overcome by sequentially overexpressing hNMNAT2 and then facilitating the uptake of Tiazofurin by folate-tethered nanoparticles, which enter cells via folate receptors.

Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Drug Carriers; Drug Resistance, Neoplasm; Fluoresceins; Folic Acid Transporters; Gene Expression; Gene Knockdown Techniques; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Isoenzymes; Nanoparticles; Nicotinamide-Nucleotide Adenylyltransferase; Protein Transport; Ribavirin; RNA, Small Interfering; Transfection

The green tea polyphenol (GTP) has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anticancer effect is not well understood. PUMA (p53-upregulated modulator of apoptosis) plays an important role in the process of apoptosis induction in a variety of human tumor cells in both p53-dependent and -independent manners. However, whether or not PUMA is involved in the process of GTP-induced apoptosis in cancer cells has not been well reported. In the present study, we treated HT-29 (mutant p53) and LoVo (wild type p53) human colorectal cancer cells with different concentrations of GTP, which led to repression of cell proliferation and induction of apoptosis in both cell lines. Meanwhile, we also observed increased PUMA expression and decreased ERK (extracellular signal-regulated kinase) activity in both of GTP-treated tumor cell lines carrying different genotypes of p53. To determine the role of PUMA in GTP-induced apoptosis, we used stable RNA interference (RNAi) to suppress PUMA expression. As a result, apoptosis was abrogated in response to GTP-treatment. We also found that suppression of ERK activity by either RNAi or its specific inhibitor significantly enhanced GTP-induced PUMA expression. All these results indicate that PUMA plays a critical role in GTP-induced apoptosis pathway in human colorectal cancer cells and can be regulated partly by ERK inactivation. Demonstration of the molecular mechanism involved in the anti-cancer effect of GTP may be useful in the therapeutic target selection for p53 deficient colorectal cancer.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Humans; Phenols; Phenotype; Polyphenols; Proto-Oncogene Proteins; RNA, Small Interfering; Tea; Time Factors; Transfection

Bile acids are implicated in colorectal carcinogenesis as evidenced by epidemiological and experimental studies. We examined whether bile acids stimulate cellular invasion of human colorectal and dog kidney epithelial cells at different stages of tumor progression. Colon PC/AA/C1, PCmsrc, and HCT-8/E11 cells and kidney MDCKT23 cells were seeded on top of collagen type I gels and invasive cells were counted after 24 h incubation. Activation of the Rac1 and RhoA small GTPases was investigated by pull-down assays. Haptotaxis was analysed with modified Boyden chambers. Lithocholic acid, chenodeoxycholic acid, cholic acid and deoxycholic acid stimulated cellular invasion of SRC- and RhoA-transformed PCmsrc and MDCKT23-RhoAV14 cells, and of HCT-8/E11 cells originating from a sporadic tumor, but were ineffective in premalignant PC/AA/C1 and MDCKT23 cells. Bile acid-stimulated invasion occurred through stimulation of haptotaxis and was dependent on the RhoA/Rho-kinase pathway and signaling cascades using protein kinase C, mitogen-activated protein kinase, and cyclooxygenase-2. Accordingly, BA-induced invasion was associated with activation of the Rac1 and RhoA GTPases and expression of the farnesoid X receptor. We conclude that bile acids stimulate invasion and haptotaxis in colorectal cancer cells via several cancer invasion signaling pathways.

Topics: Bile Acids and Salts; Colorectal Neoplasms; Cyclooxygenase 2; Dose-Response Relationship, Drug; Genes, src; Guanosine Triphosphate; Humans; Integrin beta1; Isoenzymes; Membrane Proteins; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Precancerous Conditions; Prostaglandin-Endoperoxide Synthases; rhoA GTP-Binding Protein; Tumor Cells, Cultured

Mycophenolic acid (MPA), a potent and specific inhibitor of IMP dehydrogenase, exerts its anti-mitotic action by a rapid depletion of the cellular content of guanine nucleotides. Although MPA is a potent inhibitor of GTP synthesis in the HT29 line of human colorectal adenocarcinoma cells in short-term culture, its ability to depress the cloning efficiency of these cells was found to be markedly less than against the mouse mammary carcinoma line, EMT6. In vivo, MPA is efficiently converted to the biologically inactive O-glucuronide derivative thereby limiting its effectiveness as an anti-tumor agent. Investigation of the fate of MPA incubated with monolayer cultures of HT29 and EMT6 cells revealed that the compound is rapidly converted to the O-glucuronide derivative by HT29 cells, but not by EMT6 cells. Confirmation of the identity of the glucuronide formed by HT29 cells was obtained by its conversion to MPA after incubation with beta-glucuronidase and by comparison of the mass spectrum of its HPLC peak with that of synthetic MPA O-glucuronide. Cultures of two other lines of human colorectal adenocarcinoma cells, Colo-205 and LoVo, also depleted their culture media of MPA although we have not yet established whether these cells also synthesize the glucuronide. The intrinsic partial resistance of HT29 cells to MPA appears to be associated with the ability of these cells to convert MPA to the biologically inactive glucuronide. These results, in conjunction with other reports of the capacity of colorectal cancer cells for Phase I and II metabolism of xenobiotics, may have implications for the design of drugs intended for the treatment of colorectal cancer.

Topics: Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Cell Division; Colorectal Neoplasms; Drug Resistance, Neoplasm; Female; Glucuronates; Glucuronosyltransferase; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Kinetics; Mammary Neoplasms, Experimental; Mice; Mycophenolic Acid; Tumor Cells, Cultured

Ornithine decarboxylase (ODC) activity was measured in colon adenocarcinomas and adjacent normal-appearing colon mucosa from a total of 40 patients undergoing surgical resections. The enzyme activity was measured in the presence and absence of GTP, since recent work has demonstrated a GTP-activatable form of ODC in some murine and human tumors. In general, ODC specific activity was higher in adenocarcinomas than in adjacent normal-appearing mucosa. Of greater interest, however, was the finding that 13 of 40 tumors and 3 of 40 mucosae contained a GTP-activatable form of ODC. These are minimal estimates of the proportion of tissues positive for this enzyme form, since a multiple sampling protocol indicated that expression of a GTP-activatable ODC was not uniform throughout a given tumor. Chromatographic analyses of tumor extracts revealed the presence in some tumors of multiple size forms of ODC, only some of which were activated by GTP. Enzyme kinetic data indicated that the multiple forms of ODC can have different affinities for L-ornithine and that GTP can "normalize" the aberrant kinetic properties of these forms. While there was no statistically significant correlation of the presence of a GTP-activatable ODC with stage of disease, analysis of our data revealed a positive association of a GTP-activatable ODC with tumor site; a much higher percentage of tumors of the cecum contained this ODC isoform than tumors of other colonic segments (64% versus less than or equal to 25% for other sites). These results demonstrate (a) the presence of a functionally distinct form of ODC in some human colon adenocarcinomas and (b) a distinct regional distribution of this ODC form within the colon. We suggest this alteration in a key enzyme in the growth-associated pathway of polyamine biosynthesis may play a role in colon tumor progression.

Topics: Adenocarcinoma; Colorectal Neoplasms; Enzyme Activation; Guanosine Triphosphate; Humans; Intestinal Mucosa; Kinetics; Ornithine Decarboxylase