ATF6 supports lysosomal function in tumor cells to enable ER stress-activated macroautophagy and CMA: impact on mutant TP53 expression.

The inhibition of the unfolded protein response (UPR), which usually protects cancer cells from stress, may be exploited to potentiate the cytotoxic effect of drugs inducing ER stress. However, in this study, we found that ER stress and UPR activation by thapsigargin or tunicamycin promoted the lysosomal degradation of mutant (MUT) TP53 and that the inhibition of the UPR sensor ATF6, but not of ERN1/IRE1 or EIF2AK3/PERK, counteracted such an effect. ATF6 activation was indeed required to sustain the function of lysosomes, enabling the execution of chaperone-mediated autophagy (CMA) as well as of macroautophagy, processes involved in the degradation of MUT TP53 in stressed cancer cells. At the molecular level, by pharmacological and genetic approaches, we demonstrated that the inhibition of ATF6 correlated with the activation of MTOR and with TFEB and LAMP1 downregulation in thapsigargin-treated MUT TP53 carrying cells. We hypothesize that the rescue of MUT TP53 expression by ATF6 inhibition, could further activate MTOR and maintain lysosomal dysfunction, further inhibiting MUT TP53 degradation, in a vicious circle. The findings of this study suggest that the presence of MUT TP53, which often exerts oncogenic properties, should be considered before approaching treatments combining ER stressors with ATF6 inhibitors against cancer cells, while it could represent a promising strategy against cancer cells that harbor WT TP53.

5-AZA Upregulates SOCS3 and PTPN6/SHP1, Inhibiting STAT3 and Potentiating the Effects of AG490 against Primary Effusion Lymphoma Cells.

Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism® software (version 9) and analyzed by Student's t test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine-protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells.

HSP110 Inhibition in Primary Effusion Lymphoma Cells: One Molecule, Many Pro-Survival Targets.

Heat shock proteins (HSPs) are highly expressed in cancer cells and represent a promising target in anti-cancer therapy. In this study, we investigated for the first time the expression of high-molecular-weight HSP110, belonging to the HSP70 family of proteins, in Primary Effusion Lymphoma (PEL) and explored its role in their survival. This is a rare lymphoma associated with KSHV, for which an effective therapy remains to be discovered. The results obtained from this study suggest that targeting HSP110 could be a very promising strategy against PEL, as its silencing induced lysosomal membrane permeabilization, the cleavage of BID, caspase 8 activation, downregulated c-Myc, and strongly impaired the HR and NHEJ DNA repair pathways, leading to apoptotic cell death. Since chemical inhibitors of this HSP are not commercially available yet, this study encourages a more intense search in this direction in order to discover a new potential treatment that is effective against this and likely other B cell lymphomas that are known to overexpress HSP110.

HHV-6A Infection of Papillary Thyroid Cancer Cells Induces Several Effects Related to Cancer Progression.

Recent studies have shown that thyrocytes are permissive to HHV-6A infection and that the virus may contribute to the pathogenesis of autoimmune thyroiditis. Thyroid autoimmune diseases increase the risk of papillary cancer, which is not surprising considering that chronic inflammation activates pathways that are also pro-oncogenic. Moreover, in this condition, cell proliferation is stimulated as an attempt to repair tissue damage caused by the inflammatory process. Interestingly, it has been reported that the well-differentiated papillary thyroid carcinoma (PTC), the less aggressive form of thyroid tumor, may progress to the more aggressive follicular thyroid carcinoma (FTC) and eventually to the anaplastic thyroid carcinoma (ATC), and that to such progression contributes the presence of an inflammatory/immune suppressive tumor microenvironment. In this study, we investigated whether papillary tumor cells (BCPAP) could be infected by human herpes virus-6A (HHV-6A), and if viral infection could induce effects related to cancer progression. We found that the virus dysregulated the expression of several microRNAs, such as miR-155, miR-9, and the miR-221/222 cluster, which are involved in different steps of carcinogenesis, and increased the secretion of pro-inflammatory cytokines, particularly IL-6, which may also sustain thyroid tumor cell growth and promote cancer progression. Genomic instability and the expression of PTEN, reported to act as an oncogene in mutp53-carrying cells such as BCPAP, also increased following HHV-6A-infection. These findings suggest that a ubiquitous herpesvirus such as HHV-6A, which displays a marked tropism for thyrocytes, could be involved in the progression of PTC towards more aggressive forms of thyroid tumor.

The inhibition of IRE1alpha/XBP1 axis prevents EBV-driven lymphomagenesis in NSG mice.

IMPORTANCE: The novelty of this study lies in the fact that it shows that IRE1 alpha endoribonuclease inhibition by 4µ8C was able to counteract Epstein-Barr virus-driven lymphomagenesis in NOD SCID gamma mice and prevent B-cell immortalization in vitro, unveiling that this drug may be a promising therapeutic approach to reduce the risk of post-transplant lymphoproliferative disorders (PTLD) onset in immune-deficient patients. This hypothesis is further supported by the fact that 4µ8C impaired the survival of PTLD-like cells derived from mice, meaning that it could be helpful also in the case in which there is the possibility that these malignancies have begun to arise.

EBV infection of primary colonic epithelial cells causes inflammation, DDR and autophagy dysregulation, effects that may predispose to IBD and carcinogenesis.

EBV is a gammaherpesvirus strongly associated to human cancer. The virus has been shown to play a role also in inflammatory diseases, including IBD, in the context of which colon cancer more frequently arise. In this study, we show for the first time that EBV infects primary colonic epithelial cells (HCoEpC), promotes pro-inflammatory cytokine secretion and activates molecular pathways bridging inflammation and cancer, such as ERK1/2. These effects, occurring in the course of the lytic phase of the viral life cycle, led to DDR and autophagy dysregulation. Such cellular responses, playing a key role in the maintenance of proteostasis and genome integrity, are essential to prevent carcinogenesis. Interestingly, we found that the use of the demethylating agent 5-AZA could counteract most of the effects induced by EBV infection in HCoEpC, suggesting that DNA hyper-methylation may strongly contribute to viral-driven inflammation and colon cancer predisposition.

Acetylation increases expression, interaction with TRAPPC4 and surface localization of PD-L1.

PD-L1 is an immune checkpoint inhibitor, whose surface expression may be exploited by cancer cells to escape T cell-mediated immune recognition. PD-L1 expression and nuclear localization can be affected by epigenetic modifications, such as acetylation. In this study, we showed that VPA, a class I/IIa HDAC inhibitor, upregulated PD-L1 expression on the surface of pancreatic cancer cells. To this effect contributed the increased transcription, in correlation with histone acetylation of the PD-L1 gene and the acetylation of PD-L1 protein, which led to an increased interaction with TRAPPC4, molecule involved in PD-L1 recycling to the cell membrane. Interestingly, the BRD4 inhibitor JQ-1, counteracted PD-L1 transcription and reduced its surface expression, suggesting that such a combination could improve the outcome of VPA treatment, also because it increased the cytotoxic effect of VPA. Also considering that this HDACi did not upregulate PD-L2 and that the supernatant of VPA-treated cancer cells did not increase PD-L1 expression on the surface of macrophages exposed to it.

NFE2L2 and STAT3 Converge on Common Targets to Promote Survival of Primary Lymphoma Cells.

NFE2L2 and STAT3 are key pro-survival molecules, and thus, their targeting may represent a promising anti-cancer strategy. In this study, we found that a positive feedback loop occurred between them and provided evidence that their concomitant inhibition efficiently impaired the survival of PEL cells, a rare, aggressive B cell lymphoma associated with the gammaherpesvirus KSHV and often also EBV. At the molecular level, we found that NFE2L2 and STAT3 converged in the regulation of several pro-survival molecules and in the activation of processes essential for the adaption of lymphoma cells to stress. Among those, STAT3 and NFE2L2 promoted the activation of pathways such as MAPK3/1 and MTOR that positively regulate protein synthesis, sustained the antioxidant response, expression of molecules such as MYC, BIRC5, CCND1, and HSP, and allowed DDR execution. The findings of this study suggest that the concomitant inhibition of NFE2L2 and STAT3 may be considered a therapeutic option for the treatment of this lymphoma that poorly responds to chemotherapies.

NRF2 and STAT3: friends or foes in carcinogenesis?

NRF2 is a transcription factor that plays a pivotal role in carcinogenesis, also through the interaction with several pro-survival pathways. NRF2 controls the transcription of detoxification enzymes and a variety of other molecules impinging in several key biological processes. This perspective will focus on the complex interplay of NRF2 with STAT3, another transcription factor often aberrantly activated in cancer and driving tumorigenesis as well as immune suppression. Both NRF2 and STAT3 can be regulated by ER stress/UPR activation and their cross-talk influences and is influenced by autophagy and cytokines, contributing to shape the microenvironment, and both control the execution of DDR, also by regulating the expression of HSPs. Given the importance of these transcription factors, more investigations aimed at better elucidating the outcome of their networking could help to discover new and more efficacious strategies to fight cancer.

HSPs/STAT3 Interplay Sustains DDR and Promotes Cytokine Release by Primary Effusion Lymphoma Cells.

Primary effusion lymphoma (PEL) is a rare and aggressive B-cell lymphoma, against which current therapies usually fail. In the present study, we show that targeting HSPs, such as HSP27, HSP70 and HSP90, could be an efficient strategy to reduce PEL cell survival, as it induces strong DNA damage, which correlated with an impairment of DDR. Moreover, as HSP27, HSP70 and HSP90 cross talk with STAT3, their inhibition results in STAT3 de-phosphorylation and. On the other hand, the inhibition of STAT3 may downregulate these HSPs. These findings suggest that targeting HSPs has important implications in cancer therapy, as it can reduce the release of cytokines by PEL cells, which, besides affecting their own survival, could negatively influence anti-cancer immune response.

JQ-1/bortezomib combination strongly impairs MM and PEL survival by inhibiting c-Myc and mTOR despite the activation of prosurvival mechanisms.

Multiple myeloma (MM) and primary effusion lymphoma (PEL) are two aggressive hematologic cancers against which bortezomib and JQ-1, proteasome and bromodomain and extraterminal domain (BET) inhibitors, respectively, have been shown to have a certain success. However, the combination of both seems to be more promising than the single treatments against several cancers, including MM. Indeed, in the latter, proteasome inhibition upregulated nuclear respiratory factor 1 (NRF1), and such a prosurvival effect was counteracted by BET inhibitors. In the present study, we found that JQ-1/bortezomib induced a strong cytotoxic effect against PEL and discovered new insights into the cytotoxic mechanisms induced by such a drug combination in PEL and MM cells. In particular, a stronger c-Myc downregulation, leading to increased DNA damage, was observed in these cells after treatment with JQ-1/bortezomib than after treatment with the single drugs. Such an effect contributed to mechanistic target of rapamycin (mTOR)-phosphorylated eukaryotic translation initiation factor 4E-binding protein 1 (p-4EBP1) axis inhibition, also occurring through c-Myc downregulation. However, besides the prodeath effects, JQ-1/bortezomib activated unfolded protein response (UPR) and autophagy as prosurvival mechanisms. In conclusion, this study demonstrated that JQ-1/bortezomib combination could be a promising treatment for MM and PEL, unveiling new molecular mechanisms underlying its cytotoxic effect, and suggested that UPR and autophagy inhibition could be exploited to further potentiate the cytotoxicity of JQ-1/bortezomib.

c-Myc Sustains Pancreatic Cancer Cell Survival and mutp53 Stability through the Mevalonate Pathway.

It has been shown that wild-type (wt)p53 inhibits oncogene c-Myc while mutant (mut)p53 may transactivate it, with an opposite behavior that frequently occurs in the crosstalk of wt or mutp53 with molecules/pathways promoting carcinogenesis. Even if it has been reported that mutp53 sustains c-Myc, whether c-Myc could in turn influence mutp53 expression remains to be investigated. In this study, we found that pharmacological or genetic inhibition of c-Myc downregulated mutp53, impaired cell survival and increased DNA damage in pancreatic cancer cells. At the molecular level, we observed that c-Myc inhibition reduced the expression of mevalonate kinase (MVK), a molecule belonging to the mevalonate pathway that-according to previous findings-can control mutp53 stability, and thus contributes to cancer cell survival. In conclusion, this study unveils another criminal alliance between oncogenes, such as c-Myc and mutp53, that plays a key role in oncogenesis.

Concomitant Inhibition of IRE1α/XBP1 Axis of UPR and PARP: A Promising Therapeutic Approach against c-Myc and Gammaherpesvirus-Driven B-Cell Lymphomas.

It is emerging that targeting the adaptive functions of Unfolded Protein Response (UPR) may represent a promising anti-cancer therapeutic approach. This is particularly relevant for B-cell lymphomas, characterized by a high level of constitutive stress due to high c-Myc expression. In this study, we found that IRE1α/XBP1 axis inhibition exerted a stronger cytotoxic effect compared to the inhibition of the other two UPR sensors, namely PERK and ATF6, in Burkitt lymphoma (BL) cells, in correlation with c-Myc downregulation. Interestingly, such an effect was more evident in Epstein-Barr virus (EBV)-negative BL cells or those cells expressing type I latency compared to type III latency BL cells. The other interesting finding of this study was that the inhibition of IRE1α/XBP1 downregulated BRCA-1 and RAD51 and potentiated the cytotoxicity of PARP inhibitor AZD2661 against BL cells and also against Primary Effusion Lymphoma (PEL), another aggressive B-cell lymphoma driven by c-Myc and associated with gammaherpesvirus infection. These results suggest that combining the inhibition of UPR sensors, particularly IRE1α/XBP1 axis, and molecules involved in DDR, such as PARP, could offer a new therapeutic opportunity for treating aggressive B-cell lymphomas such as BL and PEL.

ATF6 prevents DNA damage and cell death in colon cancer cells undergoing ER stress.

Colon cancer represents one of the most common and aggressive cancers in its advanced state. Among the most innovative anti-cancer approaches, the manipulation of UPR is a promising one, effective also against cancers carrying dysfunctional p53. Interestingly, it is emerging that UPR cross-talks with DDR and that targeting the interplay between these two adaptive responses may be exploited to overcome the resistance to the single DDR- and UPR-targeting treatments. Previous studies have highlighted the role of IRE1 alpha and PERK UPR sensors on DDR, while the impact of ATF6 on this process remains under-investigated. This study shows for the first time that ATF6 sustains the expression level of BRCA-1 and protects colon cancer cells from the cytotoxic effect of ER stressors DPE and Thapsigargin. At molecular level, ATF6 activates mTOR to sustain the expression of HSP90, of which BRCA-1 is a client protein. Therefore, pharmacological or genetic inhibition of ATF6 promoted BRCA-1 degradation and increased DNA damage and cell death, particularly in combination with Adriamycin. All together this study suggests that targeting ATF6 may not only potentiate the cytotoxic effect of drugs triggering ER stress but may render colon cancer cells more sensitive to Adriamycin and possibly to other DNA damaging agents used to treat colon cancer.

Mechanisms of Sensitivity and Resistance of Primary Effusion Lymphoma to Dimethyl Fumarate (DMF).

PEL is a rare B cell lymphoma associated with KSHV that mainly arises in immune-deficient individuals. The search for new drugs to treat this cancer is still ongoing given its aggressiveness and the poor response to chemotherapies. In this study, we found that DMF, a drug known for its anti-inflammatory properties which is registered for the treatment of psoriasis and relapsing-remitting MS, could be a promising therapeutic strategy against PEL. Indeed, although some mechanisms of resistance were induced, DMF activated NRF2, reduced ROS and inhibited the phosphorylation of STAT3 and the release of the pro-inflammatory and immune suppressive cytokines IL-6 and IL-10, which are known to sustain PEL survival. Interestingly, we observed that DMF displayed a stronger cytotoxic effect against fresh PEL cells in comparison to PEL cell lines, due to the activation of ERK1/2 and autophagy in the latter cells. This finding further encourages the possibility of using DMF for the treatment of PEL.

The impairment of DDR reduces XBP1s, further increasing DNA damage, and triggers autophagy via PERK/eIF2alpha in MM and IRE1alpha/JNK1/2 in PEL cells.

Cancer cells, particularly MM, that are highly secretory cells, and PEL cells that harbor KSHV, are characterized by high level of stress to which they adapt by activating DDR, UPR and autophagy. It is known that UPR sensors may affect DDR, but whether DDR manipulation influences UPR is less known. In this study, we found an intricate interplay between these responses. Indeed, PARP and CHK1 inhibition by AZD2461 and UCN-01, by downregulating c-Myc, reduced the expression of XBP1s, constitutively expressed in these cells, and upregulated CHOP. Interestingly, given the role of XBP1s in regulating DDR, BRCA-1 expression level was reduced, exacerbating DNA damage. Finally, DDR/UPR interplay activated a pro-survival autophagy via PERK/eIF2alpha axis in MM and IRE1alpha/JNK axis in PEL cells, since in the latter case PERK/eIF2alpha activation could be prevented by KSHV that, as other herpesviruses, tries to avoid the blocks of protein translation that this pathway may induce.

Targeting c-Myc Unbalances UPR towards Cell Death and Impairs DDR in Lymphoma and Multiple Myeloma Cells.

Multiple myeloma (MM) and primary effusion lymphoma (PEL) are aggressive hematological cancers, for which the search for new and more effective therapies is needed. Both cancers overexpress c-Myc and are highly dependent on this proto-oncogene for their survival. Although c-Myc inhibition has been shown to reduce PEL and MM survival, the underlying mechanisms leading to such an effect are not completely clarified. In this study, by pharmacologic inhibition and silencing, we show that c-Myc stands at the cross-road between UPR and DDR. Indeed, it plays a key role in maintaining the pro-survival function of UPR, through the IRE1α/XBP1 axis, and sustains the expression level of DDR molecules such as RAD51 and BRCA1 in MM and PEL cells. Moreover, we found that c-Myc establishes an interplay with the IRE1α/XBP1 axis whose inhibition downregulated c-Myc, skewed UPR towards cell death and enhanced DNA damage. In conclusion, this study unveils new insights into the molecular mechanisms leading to the cytotoxic effects of c-Myc inhibition and reinforces the idea that its targeting may be a promising therapeutic approach against MM and PEL that, although different cancers, share some similarities, including c-Myc overexpression, constitutive ER stress and poor response to current chemotherapies.

3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol) Counteracts ERK1/2 and mTOR Activation, Pro-Inflammatory Cytokine Release, Autophagy and Mitophagy Reduction Mediated by Benzo[a]pyrene in Primary Human Colonic Epithelial Cells.

Understanding the effects induced by carcinogens on primary colonic epithelial cells and how to counteract them might help to prevent colon cancer, which is one of the most frequent and aggressive cancers. In this study, we exposed primary human colonic epithelial cells (HCoEpC) to Benzo[a]pyrene (B[a]P) and found that it led to an increased production of pro-inflammatory cytokines and activated ERK1/2 and mTOR. These pathways are known to be involved in inflammatory bowel disease (IBD), which represents a colon cancer risk factor. Moreover, B[a]P reduced autophagy and mitophagy, processes whose dysregulation has been clearly demonstrated to predispose to cancer either by in vitro or in vivo studies. Interestingly, all the effects induced by B[a]P could be counteracted by 3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol, H), the most powerful anti-inflammatory and antioxidant compound contained in olive oil. This study sheds light on the mechanisms that could be involved in colon carcinogenesis induced by a chemical carcinogen and identifies a safe natural product that may help to prevent them.