Hypoxia attenuates the proinflammatory response in colon cancer cells by regulating IκB

Abstract

Two main features common to all solid tumors are tissue hypoxia and inflammation, both of which cause tumor progression, metastasis, therapy resistance and increased mortality. Chronic inflammation is associated with increased cancer risk, as demonstrated for inflammatory bowel disease patients developing colon cancer. However, the interplay between hypoxia and inflammation on the molecular level remains to be elucidated. We found that MC-38 mouse colon cancer cells contain functional hypoxic (HIF-1α) and inflammatory (p65/RelA) signaling pathways. In contrast to cells of the myeloid lineage, HIF-1α levels remained unaffected in MC-38 cells treated with LPS, and hypoxia failed to induce NF-κB. A similar regulation of canonical HIF and NF-κB target genes confirmed these results. RNA deep sequencing of HIF-1α and p65/RelA knock-down cells revealed that a surprisingly large fraction of HIF target genes required p65/RelA for hypoxic regulation and a number of p65/RelA target genes required HIF-1α for proinflammatory regulation, respectively. Hypoxia attenuated the inflammatory response to LPS by inhibiting nuclear translocation of p65/RelA independently of HIF-1α, which was associated with enhanced IκBα levels and decreased IKKβ phosphorylation. These data demonstrate that the interaction between hypoxic and inflammatory signaling pathways needs to be considered when designing cancer therapies targeting HIF or NF-κB.

Keywords: NF-κB; inflammatory bowel disease; lipopolysaccharide; tissue oxygenation; tumor hypoxia.

Figure 1. Hypoxic response of MC-38 cells

A. Time-dependent RT-qPCR quantification of HIF-1α and HIF-2α mRNA levels following exposure to hypoxia as indicated. B. Kinetics of hypoxic HIF-1α protein accumulation analysed by immunoblotting of 50 μg total protein extracts. C. Kinetics of hypoxic induction of canonical HIF target genes quantified by RT-qPCR of GLUT-1, CAIX and PHD3 mRNA. Shown are mean values + SD of mRNA ratios relative to the constitutively expressed ribosomal protein S12 mRNA levels of n = 3 independent experiments; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t-test).

Figure 2. Inflammatory response of MC-38 cells

A. Immunofluorescence microscopy of p65/RelA in MC-38 cells following treatment with 1 μg/ml LPS for 40 minutes. B. Immunoblot detection of p65/RelA and HDAC1 in nuclear extracts derived from MC-38 cells following stimulation with 1 μg/ml LPS for 1 hour. C. Kinetics of inflammatory induction (1 μg/ml LPS for the time periods indicated) of canonical NF-κB target genes quantified by RT-qPCR of TNF-α, IL-6 and COX-2 mRNA. Shown are mean values + SD of mRNA ratios relative to the constitutively expressed ribosomal protein S12 mRNA levels of n = 3 to 4 independent experiments; **, p < 0.01 (Student's t-test).

Figure 3. NF-κB signaling under hypoxic conditions

A. Immunofluorescence microscopy of p65/RelA in MC-38 cells following exposure to 0.2% oxygen for 0.5 to 4 hours. Treatment with 1 μg/ml LPS for 1 hour served as positive control. B. Immunoblot detection p65/RelA and the constitutive Sp1 transcription factors in MC-38 nuclear extracts following exposure to 1 μg/ml LPS or 0.2% oxygen for 1 to 12 hours. C. RT-qPCR analysis of the regulation of the canonical NF-κB target genes Tnfa, Il6 and Cox2 following hypoxic exposure.

Figure 4. HIF signaling under inflammatory conditions

A. RT-qPCR analysis of the HIF-1α mRNA response to LPS treatment in MC-38 cells. B. Kinetics of the HIF-1α protein response to LPS and/or hypoxia as assessed by immunoblotting of 50 μg total cell extracts. β-Actin served as loading control. C. RT-qPCR analysis of the effects of LPS treatment on the canonical HIF targets gene Glut1, Ca9 and Phd3.

Figure 5. HIF signaling in p65/RelA knock-down cells

A. RT-qPCR analysis of HIF-1α and p65/RelA mRNA in MC-38 cells stably transfected with shp65/RelA or shMOCK_B negative control constructs. B. Immunoblotting of HIF-1α and p65/RelA protein after 8 to 72 hours of hypoxic exposure of shMOCK_B or shp65/RelA MC-38 cells. β-Actin served as loading control. C. RT-qPCR analysis of the canonical HIF target genes Glut1, Ca9 and Phd3 in shMOCK_B or shp65/RelA MC-38 cells cultured under normoxic or hypoxic conditions as indicated.

Figure 6. p65/RelA signaling in HIF-1α knock-down cells

A. RT-qPCR analysis of HIF-1α and p65/RelA mRNA in MC-38 cells stably transfected with shHIF1α or shMOCK_A control constructs. B. Immunoblotting of HIF-1α and p65/RelA protein after 8 hours of hypoxic exposure of shMOCK_A or shHIF1α MC-38 cells. β-Actin served as loading control. C. Immunofluorescence microscopy of p65/RelA in shMOCK_A or shHIF-1α MC-38 cells treated with 1 μg/ml LPS for 40 minutes. D. RT-qPCR analysis of the canonical NF-κB target genes Tnfa and Il6 in shMOCK_A or shHIF-1α MC-38 cells upon treatment with LPS for 1 to 8 hours.

Figure 7. Interaction between inflammatory and hypoxic signaling

MC-38 shMock and shHIF-1α cells were exposed to hypoxia (0.2% oxygen) for 8 hours and treated with 1 μg/ml LPS for the last 1 hour of normoxic or hypoxic cell culture. CCL20, CXCL5, CSF2 and TNFα mRNA levels were quantified by RT-qPCR. Shown are mean values + SD of mRNA ratios relative to the constitutively expressed ribosomal protein S12 mRNA levels, normalized to the mean of each experiment (n = 6 independent experiments). Two-way ANOVA was used to assess the significance of the effects of inflammation (LPS) and hypoxia (0.2% oxygen) as well as of the interaction between these two conditions (i.e. the effect of hypoxia on inflammation). p values are indicated; n.s., not significant.

Figure 8. Inverse regulation of NF-κB and IκB under hypoxic conditions

A. Immunofluorescence microscopy of p65/RelA in shMOCK and shHIF-1α MC-38 cells following exposure to 0.2% oxygen for 8 hours and/or 1 μg/ml LPS for the last 40 minutes before harvesting. B. Co-immunofluorescence microscopy of IκBα and p65/RelA in MC-38 cells (without GFP) exposed to 0.2% oxygen for 8 hours and/or 1 μg/ml LPS for the last 30 minutes before harvesting. C. For fractional quantification of the results shown in B., at least 200 cells of each experiment were classified according to the subcellular localization of p65/RelA and the expression of IκBα as indicated. D. Immunoblot analysis of total IκBα, phosphorylated IκBα, total IKKβ and phosphorylated IKKβ/α in MC-38GFP cells exposed to 0.2% oxygen for 8 hours and 1 μg/ml LPS for the last 5, 15 or 30 minutes as indicated. E. RT-qPCR analysis of IκBα mRNA levels in MC-38GFP cells exposed to 8 hours hypoxia and/or 1 hour LPS. Shown are mean values + SD of mRNA ratios relative to the constitutively expressed ribosomal protein S12 mRNA levels, normalized to the mean of each experiment (n = 3 independent experiments); *, p < 0.05; **, p < 0.01 (Student's t-test).