Acquisition of Chemoresistance and Other Malignancy-related Features of Colorectal Cancer Cells Are Incremented by Ribosome-inactivating Stress

Abstract

Colorectal cancer (CRC) as an environmental disease is largely influenced by accumulated epithelial stress from diverse environmental causes. We are exposed to ribosome-related insults, including ribosome-inactivating stress (RIS), from the environment, dietary factors, and medicines, but their physiological impacts on the chemotherapy of CRC are not yet understood. Here we revealed the effects of RIS on chemosensitivity and other malignancy-related properties of CRC cells. First, RIS led to bidirectional inhibition of p53-macrophage inhibitory cytokine 1 (MIC-1)-mediated death responses in response to anticancer drugs by either enhancing ATF3-linked antiapoptotic signaling or intrinsically inhibiting MIC-1 and p53 expression, regardless of ATF3. Second, RIS enhanced the epithelial-mesenchymal transition and biogenesis of cancer stem-like cells in an ATF3-dependent manner. These findings indicate that gastrointestinal exposure to RIS interferes with the efficacy of chemotherapeutics, mechanistically implying that ATF3-linked malignancy and chemoresistance can be novel therapeutic targets for the treatment of environmentally aggravated cancers.

Keywords: activating transcription factor 3 (ATF3); chemoresistance; colorectal cancer; macrophage inhibitory cytokine 1 (MIC-1); ribosome-inactivating stress (RIS); toxicity; toxicology; toxin.

FIGURE 1.

Effects of RIS exposure on morphological change and resistance to anticancer drugs in suspended colon cancer cells. A, definition of “pre-exposure” and a schematic for the analysis of effects of RIS on circulating colorectal cancer cells in response to anticancer drug. Treatment of RIS-inducing agents such as DON and ANS under suspension corresponding to circulating led to anoikis-resistant states during recolonization. HCT-116 colon cancer cells were seeded in glass-bottom plates, pre-exposed to 500 ng/ml DON or 50 ng/ml ANS for 48 h, fixed, and stained with DAPI and FITC-phalloidin (dashed box). B, HCT-116 cells were exposed to serial concentrations of DON or ANS for 24 h, and changes in total protein concentration per cell (protein synthesis) were measured. Levels of total protein synthesis for 24 h in the absence or presence of the specific ribosome-directed inactivating chemical (DON or ANS) were compared, and the percentiles of protein synthesis inhibition levels per cell were calculated and statistically analyzed by linear regression. We defined the degree of RIS as the percentile of translational inhibition by the specific ribosome-directed inactivating chemicals. C, various cancer cells, including A549, HepG2, A2780, and HCT-116 cells pre-exposed to 500 ng/ml DON or 50 ng/ml for 24 h, were treated with 375 μm 5-FU for 48 h. Detached cells were collected and counted with a hemocytometer. Con, control. D, HCT-116 cells pre-exposed to 500 ng/ml DON or 50 ng/ml ANS for 24 h were treated with 375 μm 5-FU for 48 h and counted for the evaluation of survival after staining with trypan blue. E, various colon cancer cell lines pre-exposed to 500 ng/ml DON for 24 h were treated with 5-FU for 48 h and counted for the evaluation of cytotoxicity to 5-FU after staining with trypan blue. F, the nuclear fragmentation and morphology of cells were identified with DAPI and FITC-phalloidin, respectively. Nuclear fragmented cells were counted and statically analyzed by one-way analysis of variance. DMSO, dimethyl sulfoxide. G, staining with Annexin V and PI in cells pre-exposed to 500 ng/ml DON or 50 ng/ml ANS in response to 375 μm 5-FU. H, colony-forming capability of HCT-8 cells exposed to RIS in response to 50 μm 5-FU. The relative colony number was calculated as follows: (number of colony of 5-FU / 5FU + dimethyl sulfoxide, DON, or ANS) × 100 (%). Statistically significant results by unpaired t test are presented. *, p < 0.1; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

RIS exposure results in drug resistance by modulation of proapoptotic molecules. A, HCT-116, p53 knockout, and MIC-1-deficient cells were pre-exposed to increasing doses (100, 300, and 500 μg/ml) of DON or 25, 50, and 100 ng/ml of ANS for 24 h and treated with 375 μm 5-FU for 48 h. Detached cells were collected, stained with trypan blue, and counted with a hemocytometer for evaluation of cytotoxicity to 5-FU. Statistically significant results by unpaired t test are presented by repetitive experiments (***, p < 0.001). Con, control. B, HCT-116 cells pre-exposed to corresponding doses of DON or ANS for 24 h were treated with 100 μm 5-FU for 48 h. Whole cell lysates were analyzed by Western blotting with components of apoptosis-signaling molecules. hnRNP, heterogeneous nuclear ribonucleoprotein. C and E, DON-pre-exposed (500 ng/ml DON for 24 h), p53 knockout, or MIC-1-deficient HCT-116 cells were treated with 100 μm 5-FU or 100 nm paclitaxel, respectively, for 48 h and then analyzed by Western blotting with PARP1/2, MIC-1, and p53 apoptotic signaling molecules. Heterogeneous nuclear ribonucleoproteins were used as a loading control. D and F, HCT-116 cells were pre-exposed to 500 ng/ml DON for 24 h. p53 knockout cells and MIC-1-deficient cells were treated with 100 μm 5-FU (D) and 100 nm paclitaxel (F), respectively, for 24 h. Cells were fixed, and PI-positive apoptotic cells were verified by flow cytometry. G, HCT-116 cells pre-exposed to 50 ng/ml ANS for 24 h were treated with 100 nm paclitaxel for 24 h. Whole cell lysates were analyzed with MIC-1, p53, and PARP 1/2 preapoptotic components by Western blotting. H, PI-positive dead cells were verified by flow cytometry. HCT-116 cells pre-exposed to 50 ng/ml ANS for 24 h were evaluated for PI-positive apoptotic cells in response to paclitaxel.

FIGURE 3.

RIS exposure attenuates proapoptotic signaling molecules in vivo. A, allograft assay with CMT-93 cells pre-exposed to DON. CMT-93 C57BL/6 mouse colon cancer cells pre-exposed to 500 ng/ml DON for 24 h were dissociated into single cells with trypsin, and 5 × 10⁶ cells resuspended with 200 μl of PBS were injected subcutaneously into 14-week-old male C57BL/6 mice. Seven days later, 100 mg/ml 5-FU was intraperitoneally injected, and tumors were excised surgically 24 h later. The tumor volume was calculated as tumor volume = π/6 × major diameter (W) × minor diameter (L)², presented by vertical scatter plot, and statistically analyzed by unpaired two-tailed t test (bottom panel). The difference in tumor volume was statistically significant (**, p = 0.0022). Con, control. B, a histological section of allograft tumors was analyzed by immunohistochemistry with MIC-1, EGR-1, and ATF3. 3,3′-diaminobenzidine intensity versus hematoxylin was quantitatively assessed by HistoQuest software and statistically analyzed by unpaired two-tailed t test (bottom panel). **, p < 0.01; ***, p < 0.001.

FIGURE 4.

RIS exposure causes ATF3-dependent suppression of EGR-1, an apoptosis-triggering factor. A, HCT-116 cells cultured with 10% FBS and 1% P/S RPMI for 48 h were exchanged with FBS-free RPMI for 12 h, treated with 100 μm 5-FU, harvested at the corresponding time points, and evaluated for the level of EGR-1 by Western blotting. hnRNP, heterogeneous nuclear ribonucleoprotein. B and C, HCT-116 cells pre-exposed to 500 ng/ml DON for 24 h and EGR-1 stable knockdown cells by shRNA against EGR-1 were examined for proapoptotic signaling molecules in response to 100 μm 5-FU for 48 h by Western blotting (B) and PI-positive flow cytometric quantification (C). D, empty vector- or EGR-1 expression plasmid-transfected HCT-116 cells were pre-exposed to 500 ng/ml DON for 24 h and assessed for levels of PARP1/2 fragmentation, p53, and MIC-1 in response to 100 μm 5-FU for 48 h by Western blotting. E, levels of ATF3 were analyzed in the HCT-116 cells pre-exposed to 500 ng/ml DON along with 5 nm SB203580 (SB, a MAP kinase inhibitor), 5 nm SP600125 (SP, a JNK inhibitor), and 1 nm U0126, (an ERK inhibitor) for 2 h. Dotted box, inhibitory effects of SP600125 or U0126 on JNK and ERK activation in response to treatment with DON and ANS for 1 h were evaluated by Western blotting in HCT-116 cells. Con, control; Veh, vehicle. F, HCT-116 cells pre-exposed to 500 ng/ml DON for 24 h or ATF3-overexpressing cells were evaluated for levels of EGR-1 in response to 5-FU. G, control or ATF3 antisense (AS)-expressing cells pre-exposed to 500 ng/ml DON for 24 h were treated with 100 μm 5-FU for 48 h and analyzed with proapoptotic signaling molecules.

FIGURE 5.

RIS exposure induces progression of EMT and an increase in CSC population in an ATF3-dependent manner. A, histological sections of allograft tumors were analyzed by immunohistochemistry with E-cadherin, N-cadherin, and c-Myc for the assessment of EMT in allograft tumors from 500 ng/ml DON-pre-exposed CMT93 cells. 3,3′-diaminobenzidine intensity was quantitatively and statistically analyzed by HistoQuest software and unpaired t test (bottom panel). *, p < 0.05. B and C, anchorage-independent cultured spheroids of HCT-8 and ATF3 stable knockdown cells using shRNA against ATF3 were evaluated by measuring CD44- and/or CD133-positive cell populations using flow cytometry for the analysis of CSCs. DMSO, dimethyl sulfoxide.

FIGURE 6.

A proposed working model of RIS-altered chemoresistance and malignancy of circulating tumor cells. When normal intestinal epithelial cells (IECs) transformed to malignant colorectal cancer cells (CRCCs), some detached invasive cells from solid CRC intravasate into peripheral blood through lesions of the basement membrane. Exposure to RIS can exert a dual regulatory mode toward circulating tumor cells. The first is inhibition of cytotoxicity of anticancer drugs to cancer cells via direct activation of ERK-dependent ATF3-mediated signaling. ATF3 induced by exposure to RIS attenuates the EGR-1, p53, and MIC-1 signaling axis, resulting in perturbation of cancer cell death. RIS can lead to a direct translational inhibition of MIC-1 and p53 responsible for anticancer drug-induced apoptosis. The other is the enhancing effect of RIS on the malignancy of cancer cells through increases in EMT and the CSC population. As a result, chronic exposure to RIS can finally result in chemoresistance to anticancer drugs against cancer cells accompanying cancer resurgence or exacerbation after chemotherapy.