Few studies have examined associations between plasma choline metabolites and risk of colorectal cancer (CRC). Therefore, we investigated associations between plasma biomarkers of choline metabolism [choline, betaine, dimethylglycine and trimethylamine N-oxide (TMAO)] and CRC risk among postmenopausal women in a case-control study nested within the Women’s Health Initiative Observational Study. We selected 835 matched case-control pairs, and cases were further stratified by tumor site (proximal, distal, or rectal) and stage (local/regional or metastatic). CRC was assessed by self-report and confirmed by medical records over the mean 5.2y of follow-up. Baseline plasma choline metabolites were measured by liquid chromatography-tandem mass spectrometry. In multivariable-adjusted conditional logistic regression models, plasma choline tended to be positively associated with rectal cancer risk [OR (95% CI)highest vs. lowest quartile=2.44 (0.93-6.40);P-trend=0.08], while plasma betaine was inversely associated with CRC overall [0.68 (0.47-0.99);P-trend=0.01] and with local/regional tumors [0.64 (0.42-0.99);P-trend=0.009]. Notably, the plasma betaine:choline ratio was inversely associated with CRC overall [0.56 (0.39-0.82);P-trend=0.004] as well as with proximal [0.66 (0.41-1.06);P-trend=0.049], rectal [0.27 (0.10-0.78);P-trend=0.02] and local/regional [0.50 (0.33-0.76);P-trend=0.001] tumors. Finally, plasma TMAO, an oxidative derivative of choline produced by intestinal bacteria, was positively associated with rectal cancer [3.38 (1.25-9.16);P-trend=0.02] and with overall CRC risk among women with lower (vs. higher) plasma vitamin B12 levels (P-interaction=0.003). Collectively, these data suggest that alterations in choline metabolism, which may arise early in disease development, may be associated with higher risk of CRC. The positive association between plasma TMAO and CRC risk is consistent with an involvement of the gut microbiome in CRC pathogenesis.

Myeloid cells are capable of promoting or eradicating tumor cells and the nodal functions that contribute to their different roles are still obscure. Here, we show that mice with myeloid-specific genetic loss of the NF-κB pathway regulatory kinase IKKβ exhibit more rapid growth of cutaneous and lung melanoma tumors. In a BRAFV600E/PTEN−/− allograft model, IKKβ loss in macrophages reduced recruitment of myeloid cells into the tumor, lowered expression of MHC class II molecules, and enhanced production of the chemokine CCL11, thereby negatively regulating dendritic-cell maturation. Elevated serum and tissue levels of CCL11 mediated suppression of dendritic-cell differentiation/maturation within the tumor microenvironment, skewing it toward a Th2 immune response and impairing CD8+ T cell–mediated tumor cell lysis. Depleting macrophages or CD8+ T cells in mice with wild-type IKKβ myeloid cells enhanced tumor growth, where the myeloid cell response was used to mediate antitumor immunity against melanoma tumors (with less dependency on a CD8+ T-cell response). In contrast, myeloid cells deficient in IKKβ were compromised in tumor cell lysis, based on their reduced ability to phagocytize and digest tumor cells. Thus, mice with continuous IKKβ signaling in myeloid-lineage cells (IKKβCA) exhibited enhanced antitumor immunity and reduced melanoma outgrowth. Collectively, our results illuminate new mechanisms through which NF-κB signaling in myeloid cells promotes innate tumor surveillance. Cancer Res; 74(24); 1–11. ©2014 AACR.

Myeloid cells are effectors of both anti-tumor and pro-tumor immune responses, but much needs to be determined as to signals that determine which function of the myeloid lineage dominates. Shown here, mice with myeloid-specific IKKβ loss exhibit more rapid growth of cutaneous and lung melanoma tumors. Specifically, in a BRAFV600EPTEN-/- allograft model, IKKβ loss in macrophages resulted in reduced recruitment of myeloid cells into the tumor, reduced expression of MHCII, and enhanced production of the chemokine, CCL11, which negatively regulated dendritic cell maturation. The elevated serum and tissue levels of CCL11 mediated suppression of dendritic cell differentiation/maturation within the TME, resulted in a Th2 skew of the immune response, and impaired CD8+T cell-mediated tumor cell killing. Macrophage depletion or CD8+T cell depletion in mice with IKKβWT myeloid cells enhanced tumor growth in the melanoma allograft. In contrast, mice with IKKβWT myeloid cells used the myeloid cell response to mediate anti-tumor immunity against the syngeneic B16 melanoma, with less apparent dependency on a CD8+T cell response. Myeloid cells deficient in IKKβ were compromised in tumor cell killing based upon reduced ability to phagocytize and digest tumor cells. Conversely, mice with continuous IKKβ signaling in myeloid-lineage cells (IKKβCA) exhibited enhanced anti-tumor immunity and reduced B16 melanoma tumor growth. Collectively, these data uncover new mechanisms by which NF-κB signaling in myeloid cells promotes innate tumor surveillance.

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells in which the G1–S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2–M checkpoints mediated by the ATR (ataxia telangiectasia and Rad3 related) and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR–Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs. Cancer Res; 74(24); 1–12. ©2014 AACR.

Resistance to DNA-damaging chemotherapy is a barrier to effective treatment that appears to be augmented by p53 functional deficiency in many cancers. In p53-deficient cells where the G1/S checkpoint is compromised, cell viability after DNA damage relies upon intact intra-S and G2/M checkpoints mediated by the ATR and Chk1 kinases. Thus, a logical rationale to sensitize p53-deficient cancers to DNA-damaging chemotherapy is through the use of ATP-competitive inhibitors of ATR or Chk1. To discover small molecules that may act on uncharacterized components of the ATR pathway, we performed a phenotype-based screen of 9,195 compounds for their ability to inhibit hydroxyurea-induced phosphorylation of Ser345 on Chk1, known to be a critical ATR substrate. This effort led to the identification of four small-molecule compounds, three of which were derived from known bioactive library (anthothecol, dihydrocelastryl, and erysolin) and one of which was a novel synthetic compound termed MARPIN. These compounds all inhibited ATR-selective phosphorylation and sensitized p53-deficient cancer cells to DNA-damaging agents in vitro and in vivo. Notably, these compounds did not inhibit ATR catalytic activity in vitro, unlike typical ATP-competitive inhibitors, but acted in a mechanistically distinct manner to disable ATR-Chk1 function. Our results highlight a set of novel molecular probes to further elucidate druggable mechanisms to improve cancer therapeutic responses produced by DNA-damaging drugs.