A Switch from Canonical to Noncanonical Wnt Signaling Mediates Drug Resistance in Colon Cancer Cells

Butyrate, a fermentation product of fiber in the colon, acts as a histone deacetylase inhibitor (HDACi) and induces apoptosis in colon cancer (CC) cells in vitro. We have reported that the apoptotic effects of butyrate are dependent upon the hyperactivation of the Wnt/beta-catenin pathway. However, prolonged exposure of CC cells to increasing concentrations of butyrate results in the acquisition of resistance to the Wnt/beta-catenin- and apoptosis-inducing effects of this agent, as well as cross-resistance to structurally different HDACis. Here we report that one mechanism whereby HDACi resistance arises is through the increase of beta-catenin-independent (noncanonical) Wnt signaling. Compared to HDACi-sensitive HCT-116 CC cells, HDACi-resistant HCT-R cells exhibit higher levels of AKT/PKB cell survival signaling, which is in part induced by WNT5A and its receptor ROR2. The induction of AKT signaling by HDACis is also detected in other CC cell lines, albeit to a lesser extent than in the drug-resistant HCT-R cells. The observations suggested that the apoptotic effect of butyrate and other HDACis in CC cells can be augmented by inhibitors of pAKT. In agreement with the hypothesis, the combination of MK2206, a pAKT inhibitor, and a HDACi (butyrate or LBH589) induced higher apoptosis in CC cells compared to each agent alone. The exposure to both agents also re-sensitized the HCT-R cells to apoptosis. Finally, the concept of simultaneously inducing canonical Wnt activity and suppressing AKT signaling was translated into a combination of diet-derived agents. Diet-derived pAKT inhibitors (caffeic acid phethyl ester, sulforaphane, dilallyl trisulfide) suppressed the butyrate-induced levels of pAKT, and increased the apoptotic effects of butyrate in both drug-sensitive and drug-resistant CC cells

In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

Although neoplastic cells exhibit relatively higher sensitivity to hyperthermia than normal cells, hyperthermia has had variable success as an anti-cancer therapy. This variable outcome might be due to the fact that cancer cells themselves have differential degrees of sensitivity to high temperature. We hypothesized that the varying sensitivity of colorectal cancer (CRC) cells to hyperthermia depends upon the differential induction of survival pathways. Screening of such pathways revealed that Extracellular Signal-Regulated Kinase (ERK) signaling is augmented by hyperthermia, and the extent of this modulation correlates with the mutation status of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Through clonal growth assays, apoptotic analyses and transcription reporter assays of CRC cells that differ only in KRAS mutation status we established that mutant KRAS cells are more sensitive to hyperthermia, as they exhibit sustained ERK signaling hyperactivation and increased Wingless/Integrated (WNT)/beta-catenin signaling. We propose that whereas increased levels of WNT and ERK signaling and a positive feedback between the two pathways is a major obstacle in anti-cancer therapy today, under hyperthermia the hyperinduction of the pathways and their positive crosstalk contribute to CRC cell death. Ascertaining the causative association between types of mutations and hyperthermia sensitivity may allow for a mutation profile-guided application of hyperthermia as an anti-cancer therapy. Since KRAS and WNT signaling mutations are prevalent in CRC, our results suggest that hyperthermia-based therapy might benefit a significant number, but not all, CRC patients

Comprehensive suppression of all apoptosis-induced proliferation pathways as a proposed approach to colorectal cancer prevention and therapy.

Mutations in the WNT/beta-catenin pathway are present in the majority of all sporadic colorectal cancers (CRCs), and histone deacetylase inhibitors induce apoptosis in CRC cells with such mutations. This apoptosis is counteracted by (1) the signaling heterogeneity of CRC cell populations, and (2) the survival pathways induced by mitogens secreted from apoptotic cells. The phenomena of signaling heterogeneity and apoptosis-induced survival constitute the immediate mechanisms of resistance to histone deacetylase inhibitors, and probably other chemotherapeutic agents. We explored the strategy of augmenting CRC cell death by inhibiting all survival pathways induced by the pro-apoptotic agent LBH589, a histone deacetylase inhibitor: AKT, JAK/STAT, and ERK signaling. The apoptosis-enhancing ability of a cocktail of synthetic inhibitors of proliferation was compared to the effects of the natural product propolis. We utilized colorectal adenoma, drug-sensitive and drug-resistant colorectal carcinoma cells to evaluate the apoptotic potential of the combination treatments. The results suggest that an effective approach to CRC combination therapy is to combine apoptosis-inducing drugs (e.g., histone deacetylase inhibitors, such as LBH589) with agents that suppress all compensatory survival pathways induced during apoptosis (such as the cocktail of inhibitors of apoptosis-associated proliferation). The same paradigm can be applied to a CRC prevention approach, as the apoptotic effect of butyrate, a diet-derived histone deacetylase inhibitor, is augmented by other dietary agents that modulate survival pathways (e.g., propolis and coffee extract). Thus, dietary supplements composed by fermentable fiber, propolis, and coffee extract may effectively counteract neoplastic growth in the colon

Role for ERK signaling in the apoptosis of CRC cells exposed to dietary agents and clonal growth ability of neoplastic and normal cells exposed to such agents.

<p>A. HCT116 cells were exposed for 24 h to mock (M), 1 mg/ml roasted coffee extract (R), 5 mM butyrate and 100 µg/ml propolis (BP), or butyrate/propolis and increasing concentrations of coffee extract: 0.25, 0.5 or 1.0 mg/ml. Apoptosis was measured by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#s2" target="_blank">Materials and Methods</a>. B. HCT-116 cells were treated as in (A) for 17 hours and total cell lysates were analyzed by western blotting. C. HCT-116 and LT 97 cells were exposed for 20 h, and HCT-R cells were exposed for 42 h to mock (M), 0.5 µM AZD6244 (A), the combination; 5 mM butyrate, 100 µg/ml propolis, 1 mg/ml roasted coffee extract (BPR), or BPR and 0.5 µM AZD6244 (BPR-A). Apoptosis was measured by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#s2" target="_blank">Materials and Methods</a>. Statistically significant differences in apoptotic levels are noted by asterisks (P<0.05). D. Clonal growth assays. Percent clonal growth was calculated by dividing the number of cell colonies by the number of plated cells, and multiplying by 100. The ratios of clonal growth were calculated by dividing the percent clonal growth in mock-treated samples by the percent clonal growth in agent-treated samples. Experiments were repeated four to six times with triplicate samples per experiment. Statistically significant differences among cell lines in their mean ratios were determined by one-way ANOVA. For cells exposed to butyrate, F(6,31) = 10.26, P<0.0001; for cells exposed to propolis and coffee extract, F(6,28) = 5.493, P = 0.0007, and for cells exposed to butyrate, propolis and coffee extract, F(6,26) = 10.56, P<0.0001. The post-test calculations used the Bonferroni correction to adjust for multiple comparisons with 95% confidence. Among the cells exposed to butyrate, statistically significant differences (P<0.05, CI 95%) were detected in HCT116 vs. CCL92, HCT116 vs. HEK293, HCT116 vs. SW13, HCT116 vs. LA1-5s, HCT116 vs. HCT-R, CCD841CoN vs. HEK293, and CCD841CoN vs. HCT-R cells. Among the cell lines exposed to propolis and coffee extract, statistically significant differences (P<0.05, CI 95%) were detected in HCT116 vs. SW13, HCT-R vs. CCL92, CCD841CoN vs. HCT-R cells, HEK293 vs. HCT-R, and SW13 vs. HCT-R cells. Among cell lines exposed to butyrate, propolis, and coffee extract statistically significant differences (P<0.05, CI 95%) were detected in HEK293 vs. SW13 cells, and HCT116 vs. all other cell lines except for HCT-R cell line.</p

The combination treatment of butyrate, propolis, and coffee extract induces highest levels of apoptosis in colon adenoma and carcinoma cells.

<p>A–C. Cells were exposed to mock (M), 5 mM butyrate (B), 1 mg/ml roasted coffee extract (R), 100 µg/ml propolis (P), butyrate and coffee extract (BR), butyrate and propolis (BP), propolis and coffee extract (PR), or butyrate, propolis and coffee extract (BPR). Apoptosis was measured by flow cytometry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115068#s2" target="_blank">Materials and Methods</a>. D–F. Cells were exposed to treatments as described above for 17 h, and total cell lysates were analyzed by western blotting.</p

Ability of ICAP and propolis to augment chemotherapeutics-induced apoptosis.

<p>A. HCT-R or HCT-116 cells were exposed for 48 h or 24 h respectively, to mock (M), 50 nM LBH589 (L), LBH589 and 100 µg/ml propolis (LP), LBH589 and the ICAP (LC), 100 µg/ml propolis (P), or the ICAP alone (C). B. HCT-R cells were exposed to the treatments described in A for 17 h. and total cell lysates were analyzed by western blotting. C. HCT-R and HCT-116 cells were exposed for 48 h or 24 h respectively, to mock (M), 10 µM 5-fluorouracil (F), 5-fluorouracil and 100 µg/ml propolis (FP), 5-fluorouracil and the ICAP (FC), propolis (P), or the ICAP alone (C).</p

LBH589-induced apoptosis is augmented by suppressing three survival pathways.

<p>A. Western blot analyses of HCT-R CRC cells exposed to mock (C) treatment, 50 nM LBH589 (L), 1 µM MK2206 (M), 0.5 µM AZD6244 (A), or 1 µM pyridone 6 (P) for 20 hours. Equal number of cells were lysed directly in Laemmli buffer and analyzed for expression levels of phosphorylated and total levels of AKT, ERK1/2, and STAT3. B. Apoptotic analyses of HCT-R cells exposed for 48 h to the treatments described in (A). Asterisk indicates statistically significant differences (P<0.05) between the apoptotic levels. Additional statistically significant differences are indicated in the text.</p