bims-istrec Biomed News
on Integrated stress response in cancer
Issue of 2021‒10‒31
eleven papers selected by
Vincenzo Ciminale’s Lab
Istituto Oncologico Veneto

  1. Front Cell Dev Biol. 2021 ;9 736350
      Colorectal cancer (CRC) is one of the most common malignancies in the world and has a poor prognosis. In the present research, gambogenic acid (GNA), isolated from the traditional Chinese medicine gamboge, markedly induced apoptosis and inhibited the proliferation of CRC in vitro and in vivo. Furthermore, GNA triggered endoplasmic reticulum (ER) stress, which subsequently activated inositol-requiring enzyme (IRE) 1α and the eukaryotic translation initiation factor (eIF) 2α pathway. Pretreatment with salubrinal (an eIF2α inhibitor) rescued GNA-induced cell death. Furthermore, GNA downregulated the expression of Aurora A. The Aurora A inhibitor alisertib decreased ER stress. In human colorectal adenocarcinoma tissue, Aurora A was upregulated compared to normal colorectal epithelial nuclei. Furthermore, GNA ameliorated mouse colitis-associated cancer models. Our findings demonstrated that GNA significantly inhibited the proliferation of CRC through activation of ER stress by regulating Aurora A, which indicates the potential of GNA for preventing the progression of CRC.
    Keywords:  Aurora A; colorectal cancer; endoplasmic reticulum stress; eukaryotic translation initiation factor 2α; gambogenic acid
  2. Cancer Biol Ther. 2021 Oct 25. 1-10
      ONC201 demonstrated promising activity in patients with advanced endometrial cancer in a Phase I clinical trial. ONC201 activates the integrated stress response (ISR) and upregulates TRAIL and its receptor DR5. We hypothesized ONC201 upregulation of DR5 could sensitize tumors to TRAIL and combination of ONC201 and TRAIL would lead to enhanced cell death in endometrial cancer models. Five endometrial cancer cell lines AN3CA, HEC1A, Ishikawa, RL952, and KLE as well as a murine xenograft model were treated with ONC201 alone or in combination with TRAIL. ONC201 decreased the cell viability of all five endometrial cancer cell lines at clinically achievable low micro-molar concentrations (2-4 μM). ONC201 activated the ISR and induced protein expression of TRAIL and DR5 at the cell surface. Pretreatment with ONC201 sensitized endometrial cancer cell lines to TRAIL, leading to increased cell death induction compared to either agent alone. Tumor growth was reduced in vivo by the ONC201/TRAIL combination treatment in the xenograft model of endometrial cancer (p = .014). Mice treated with combination treatment survived significantly longer than mice from the three control groups (p = .018). ONC201 decreased cell viability in endometrial cancer cells lines primarily through growth arrest while the combination of ONC201 and TRAIL promoted cell death in vitro and in vivo. Our results suggest a novel cancer therapeutic strategy that can be further investigated in the clinic.
    Keywords:  ONC201; apoptosis; death receptors; endometrial cancer; trail
  3. Toxicol Lett. 2021 Oct 20. pii: S0378-4274(21)00860-2. [Epub ahead of print]
      In this study, we aimed to elucidate the role of chronic cadmium (Cd) exposure in epithelial-mesenchymal transition (EMT) and thus malignant phenotypic changes of prostate cancer cells. Prostate cancer cells (PC-3 and DU145) were exposed to a non-toxic level (0.5 or 2 μM) of Cd for up to 3 months, which resulted in significantly promoted migration and invasion of the cells. These phenotypic changes were considered to be the consequence of enhanced EMT as evidenced by diminished expression of E-cadherin and increased vimentin expression. Regarding the mechanisms of Cd-induced EMT, we found Smad3 was activated but without upregulation of TGF-β. Alternatively, we found endoplasmic reticulum (ER) stress of prostate cancer cells was significantly evoked, which was parallel with the increased reactive oxygen species (ROS). Removal of ROS by N-acetylcysteine significantly reduced ER stress in prostate cancer cells, followed by the decrease of Smad3 phosphorylation and expression of nuclear Snail, resulting in the inhibition of EMT and malignant phenotypic changes of prostate cancer cells. These findings indicated a new TGF-β independent, ROS-mediated ER stress/Smad signaling pathway in chronic Cd exposure-induced EMT of prostate cancer cells, which could be a novel mechanism involved in cadmium-mediated cancer cells malignant transformation. Accordingly, ROS-induced ERs may become a novel preventive and therapeutic target for cancer.
    Keywords:  TGF-β-independent/Smad3 pathway; cadmium; endoplasmic reticulum stress; epithelial-mesenchymal transition; reactive oxygen species
  4. Cancer Lett. 2021 Oct 20. pii: S0304-3835(21)00534-6. [Epub ahead of print]524 161-171
      Sorafenib and its derivative regorafenib are the first- and second-line targeted drugs for advanced HCC, respectively. Although both drugs improve overall survival, drug resistance remains the major barrier to their full efficacy. Thus, strategies to enhance sorafenib and regorafenib efficacy against HCC are solely needed. Interleukin-6 receptor alpha (IL-6Rα) is the receptor of IL-6, a multi-functional cytokine, which plays key roles in liver-regeneration, inflammation and development of hepatocellular carcinoma (HCC). Here we show the expression of IL-6Rα was induced in response to sorafenib. Depletion of IL-6Rα abolished IL-6 induced STAT3 phosphorylation at 705th tyrosine and tumor growth of HCC cells under sorafenib treatment. Mechanistically, activating transcription factor 3 (ATF3) was induced in response to sorafenib and subsequently bound to the promoter of IL-6Rα, leading to its transcriptional activation. Depletion of ATF3 or its upstream transcription factor, ATF4, attenuated IL-6Rα induction and IL-6 mediated sorafenib resistance. The ATF4-ATF3-IL-6Rα cascade is also activated by regorafenib. Furthermore, blockade of IL-6Rα with the FDA approved IL-6Rα antibody drug, Sarilumab, drastically attenuated both sorafenib and regorafenib resistance in patient-derived xenograft (PDX) tumors, where human IL-6 could be detected by a novel in situ hybridization technique, named RNAscope. Together, our data reveal that ATF3-mediated IL-6Rα up-regulation promotes both sorafenib and regorafenib resistance in HCC, and targeting IL-6Rα represents a novel therapeutic strategy to enhance sorafenib/regorafenib efficacy for advanced HCC treatment.
    Keywords:  ATF3; HCC; IL-6Rα; Regorafenib; Sorafenib
  5. Cell Death Dis. 2021 Oct 29. 12(11): 1029
      Breast cancer is a major threat to women's health and estrogen receptor-positive (ER+) breast cancer exhibits the highest incidence among these cancers. As the primary estrogen, estradiol strongly promotes cellular proliferation and radiotherapy, as a standard treatment, exerts an excellent therapeutic effect on ER+ breast cancer. Therefore, we herein wished to explore the mechanism(s) underlying the inhibitory effects of radiation on the proliferation of ER+ breast cancer cells. We used the ER+ breast cancer cell lines MCF7 and T47D, and their complementary tamoxifen-resistant cell lines in our study. The aforementioned cells were irradiated at different doses of X-rays with or without exogenous estradiol. CCK8 and clone-formation assays were used to detect cellular proliferation, enzyme-linked immunosorbent assay (ELISA) to determine estradiol secretion, western immunoblotting analysis and quantitative real-time PCR to evaluate the expression of proteins, and immunofluorescence to track endoplasmic reticulum stress-related processes. Finally, BALB/C tumor-bearing nude mice were irradiated with X-rays to explore the protein expression in tumors using immunohistochemistry. We found that ionizing radiation significantly reduced the phosphorylation of estrogen receptors and the secretion of estradiol by ER+ breast cancer cells. CYP19A (aromatase) is an enzyme located in the endoplasmic reticulum, which plays a critical role in estradiol synthesis (aromatization), and we further demonstrated that ionizing radiation could induce endoplasmic reticulum stress with or without exogenous estradiol supplementation, and that it downregulated the expression of CYP19A through ER-phagy. In addition, ionizing radiation also promoted lysosomal degradation of CYP19A, reduced estradiol synthesis, and inhibited the proliferation of tamoxifen-resistant ER+ breast cancer cells. We concluded that ionizing radiation downregulated the expression of CYP19A and reduced estradiol synthesis by inducing endoplasmic reticulum stress in ER+ breast cancer cells, thereby ultimately inhibiting cellular proliferation.
  6. Mol Biol Rep. 2021 Oct 26.
      BACKGROUND: The long-term use of dexamethasone (Dex), a well-known immunosuppressant, leads to an imbalance in bone metabolism and rapid decline of bone mineral density due to apoptosis of osteoblasts. The molecular mechanisms by which Dex induces osteoblast apoptosis remain unclear.MATERIALS AND METHODS: MC3T3-E1 cells were treated with 0, 10-8, 10-6, and 10-4 M Dex for 24 h. ATF6, phosphorylated PERK, PERK, phosphorylated IRE1, and IRE1 expression, cell apoptosis, and caspase-12 and caspase-3 activity were measured. CHOP expression and calcium ion influx rate were measured in cells treated with 0 and 10-4 M Dex for 24 h. The effect of 2-APB treatment was assessed in cells treated with 0 or 10-4 M Dex.
    RESULTS: Levels of ATF6 and phosphorylated PERK and IRE1 increased in a dose-dependent manner in MC3T3-E1 cells treated with 10-8, 10-6, and 10-4 M Dex, compared to the control group (P < 0.05). Cells treated with 10-6 and 10-4 M Dex had significantly increased apoptotic rates and caspase-12 and caspase-3 activities (P < 0.05). Cells treated with 10-4 M Dex had significantly increased CHOP levels and calcium ion influx rates (P < 0.05). Combined treatment with 10-4 M Dex and 2-APB abrogated the observed increases in cell apoptosis and caspase-12 and caspase-3 activities (P < 0.05).
    CONCLUSIONS: High doses of Dex induce CHOP expression by promoting calcium ion influx-dependent induction of ATF6, phosphorylated PERK and phosphorylated IRE1, which induce endoplasmic reticulum stress-mediated apoptosis in osteoblasts. 2-APB protects the osteoblasts from the effects of Dex, preventing endoplasmic reticulum stress-mediated apoptosis.
    Keywords:  Apoptosis; CHOP; Dexamethasone; Endoplasmic reticulum stress; Osteoblasts; Osteoporosis
  7. Clin Transl Med. 2021 Oct;11(10): e587
      Chemoresistance remains a major obstacle to successful cancer therapy, especially for advanced cancers. It used to be recognised as a stable outcome resulting from genetic changes. However, recent studies showed that chemoresistance can also be unstable and reversible with the involvement of non-genetic alterations. In the present study, we found that activating transcription factor 4 (ATF4) is downregulated in chemoresistant gastric cancer cells. The over-expression of ATF4 reversed chemoresistance by activating CHOP transcription to enhance drug-induced apoptosis, and vice versa. Moreover, casein kinase 1 delta (CK1δ) was identified as the kinase responsible for ATF4-S219 phosphorylation, which triggered βTrCP-mediated ATF4 polyubiquitination to promote its proteasomal degradation subsequently. Interestingly, drug withdrawal gradually restored chemosensitivity as well as ATF4 expression in chemoresistant cells, highlighting the dependence of dynamic drug resistance on ATF4 protein expression. In line with these findings, the inhibition of ATF4 protein degradation by CK1δ or proteasome inhibitors overcame chemoresistance both in vitro and in vivo. Taken together, these results indicate that CK1δ stimulates βTrCP-dependent ATF4 polyubiquitination and subsequent proteasomal degradation to promote chemoresistance in gastric cancer. Stabilisation of the ATF4 protein with bortezomib (BTZ), an anticancer drug that inhibits proteasomal degradation, might be a rational strategy to improve chemotherapeutic efficacy in gastric cancer.
    Keywords:  ATF4; CK1δ; chemoresistance; gastric cancer; phosphorylation; ubiquitination
  8. Int J Radiat Oncol Biol Phys. 2021 Nov 01. pii: S0360-3016(21)01248-7. [Epub ahead of print]111(3S): e47-e48
      PURPOSE/OBJECTIVE(S): Hepatocellular carcinoma (HCC) is a highly malignant liver cancer and is one of the leading causes of cancer-related mortality worldwide. Sorafenib, a multikinase inhibitor, is the main first-line treatment for advanced HCC, yet its survival benefits are limited. Tumor Treating Fields (TTFields) therapy is an antimitotic treatment, in which low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields are delivered to tumors non-invasively and loco-regionally. This research aimed to evaluate the efficacy of TTFields for the treatment of HCC in vitro and in vivo, alone and in combination with sorafenib, and to elucidate the underlying mechanism of action.MATERIALS/METHODS: HCC cell lines (HepG2 and Huh-7D12) were treated with various frequencies of TTFields to identify which is the most cytotoxic frequency. The effect of TTFields at optimal frequency in combination with sorafenib was tested. Cytotoxic and apoptotic effects were examined, as well as ER stress levels and responsive pathways. In vivo, N1S1 HCC cells (50,000) were orthotopically injected into the left hepatic lobe of SD rats, and tumors allowed to develop for 7 days. TTFields (at optimal frequency) or sham were then continuously applied for 6 days to the abdominal region of the rats, with concomitant sorafenib (10 mg/kg/day) or vehicle injected daily. Tumor volume growth was determined by MRI, and levels of cellular death were examined by immunohistochemistry.
    RESULTS: TTFields were found to be most cytotoxic to HCC cells at 150 kHz. Cells displayed dose-dependent sensitivity to sorafenib, and concomitant delivery of 150 kHz TTFields augmented this effect. Each modality alone led to increased levels of autophagy induced by elevated ER stress. The combination of TTFields and sorafenib triggered stress responsive pathways resulting in increased cellular death. In the animal model, tumor growth was significantly reduced and levels of cleaved PARP were elevated, in the combination group compared to other treatment groups, indicative of increased apoptosis.
    CONCLUSION: The results demonstrate that TTFields and sorafenib each induce ER stress leading to autophagy in HCC, and when combined, the elevated stress increases cellular death, both in vitro and in vivo. The ongoing phase 2 HEPANOVA (NCT03606590) clinical trial will investigate the safety and efficacy of TTFields plus sorafenib in patients with unresectable, locally advanced HCC.
  9. Int J Radiat Oncol Biol Phys. 2021 Nov 01. pii: S0360-3016(21)01711-9. [Epub ahead of print]111(3S): e253
      PURPOSE/OBJECTIVE(S): Onc201 is a novel therapeutic in clinical trials that displays widespread anti-cancer activity and also has been shown to act as a radiosensitizer. Previously, we showed that Onc201 and its derivatives activate a mitochondrial protease, ClpP, resulting in mitochondrial protein degradation, induction of ATF4/CHOP and cytostatic activity in a breast cancer cell line. However, how activation of ClpP, a mitochondrial protease, signals to the nucleus has not been established. We show here that Onc201 induces GDF15, a mitochondrial stress cytokine, and further delineate the upstream signaling pathways. The aim of these studies is to better understand mechanisms of mitochondrial to nuclear signaling to allow for more effective anti-cancer strategies to be developed.MATERIALS/METHODS: To monitor mitochondrial to nuclear signaling, Sum159, a breast cancer cell line, was treated with Onc201 or its derivatives, and monitored for ATF4 or GDF15 protein expression by Western blotting. Delineation of the signaling pathways involved was achieved using siRNA directed against various targets or by use of specific kinase inhibitors.
    RESULTS: Treatment of Sum159 cells with Onc201 resulted in the induction of GDF15 and ATF4 protein as early as ∼17 hours after treatment. Using siRNA knockdowns, we found that GDF15 induction is dependent upon ClpP, ATF4, and CHOP. However, knockdown of GCN2 or HRI, which are eiF2a kinases known to regulate ATF4, had no effect on GDF15, nor did siRNA against IRE1a. Because we found that Onc201 induced Erk, Akt and AMPK activity, we targeted these pathways with specific kinase inhibitors; however, none of these pathways were required for GDF15 induction. These results suggest that other pathways may be involved in the induction of ATF4 and GDF15 and these are currently under investigation.
    CONCLUSION: We and others have shown that Onc201 and related molecules, activate ClpP, a mitochondrial protease resulting in rapid mitochondrial protein degradation, loss of mitochondrial function, and cytostasis or apoptosis in a variety of cancer cell lines. Importantly, Onc201 shows very low toxicity to normal cell lines and is well tolerated in patients. However, additional stratagems need to be explored to improve the efficacy of Onc201. To this end, we sought to understand how mitochondrial to nuclear signaling occurs as a way to target this pathway. These results establish that ATF4/CHOP signaling is induced and this subsequently signals to induce GDF15. However, it still remains unclear how ATF4/CHOP expression is induced and this area is currently being investigated.
  10. Free Radic Biol Med. 2021 Oct 21. pii: S0891-5849(21)00773-5. [Epub ahead of print]177 156-166
      Ferroptosis, a type of programmed cell death caused by lipid peroxidation has recently been observed in colitis. Whether a high-fat diet (HFD) affects ferroptosis and whether it contributes to colitis-associated carcinogenesis (CAC) has not been explored. We found iron, lipid peroxidation, and ferroptotic markers to be elevated in AOM/DSS (azoxymethane/dextran sulfate sodium)-induced mouse CAC model. Transmission electron microscopy also confirmed the occurrence of ferroptosis in colonic tissues. Treatment with the ferroptosis inhibitor, ferrostatin-1 increased the incidence of CAC. Compared with iso-caloric control mice, HFD mice exhibited increased tumor number and a higher degree of dysplasia following repression of lipid peroxidation and ferroptosis marker expression in mouse colon tissue. Furthermore, ferroptosis markers were negatively correlated with the tumor number in mice. In vitro, a lipid mixture blocked ferroptosis in various colorectal cancer cell lines and inhibited GSH degradation by negatively regulating CHAC1, a target in ER stress signaling. Finally, the ferroptosis inducer partly abolished the pro-tumor effect of the HFD on CAC in vivo. Collectively, these findings suggest that a HFD aggravates CAC through the evasion of ferroptosis in the ER stress-mediated pathway and provide a new perspective for CAC prevention in the future.
    Keywords:  CHAC1; Colitis-associated carcinogenesis; Endoplasmic reticulum stress; Ferroptosis; High-fat diet
  11. Int J Biol Macromol. 2021 Oct 20. pii: S0141-8130(21)02257-1. [Epub ahead of print]
      Activating transcription factor 3 (ATF3), an inducible stress gene, is stimulated by transforming growth factor-beta1 (TGF-β1) in a protracted and relentless manner in human mammary cancer cells (hBC cells; MDA-MB231). The molecular mechanism behind this stable expression of ATF3 via TGF-β1 in MDA-MB231 cells is unknown. This study found that TGF-β1 stimulated the expression of the nuclear factor of activated T Cells 2 (NFATC2) in MDA-MB231 cells and provided evidence of its interaction with ATF3. The functional characterization of NFATC2 in association with ATF3 was determined by silencing of NFATC2 using siRNA. Knock-down of NFATC2 decreased the expression of both ATF3 and its target gene MMP13 (matrix metalloproteinase 13, a critical invasive gene) in hBC cells. Chromatin immunoprecipitation revealed that TGF-β1 promoted NFATC2 binding and NFATC2-ATF3 complex binding at the MMP13 promoter region, whereas silencing of NFATC2 decreased their binding in hBC cells. Thus, we uncovered the mechanism of interaction between NFATC2 and ATF3 regulated by TGF-β1, and NFATC2 acted as a pivotal factor in providing ATF3 stability and further drove MMP13 transcription. Targeting NFATC2 and blocking its association with ATF3 could therefore help to slow the progression of breast cancer.
    Keywords:  ATF3; NFATC2; TGF-β1