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Understand Tumor Immunity and Pyroptosis to find the anti-cancer strategy
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Understand Tumor Immunity and Pyroptosis to find the anti-cancer strategy.
Tumor resistance to apoptosis and an immunosuppressive tumor microenvironment are two major reasons for poor tumor response to therapy.
Pyroptosis is a lytic and inflammatory programmed cell death pathway distinct from apoptosis, and recent evidence suggests that induction of pyroptosis in tumor cells results in a strong inflammatory response and dramatic tumor regression.
Underlying its antitumor effect, pyroptosis is mediated by the pore-forming gasdermin protein, which promotes immune cell activation and infiltration through the release of pro-inflammatory cytokines and immunogenic substances after cell rupture.
However, given its inflammatory nature, aberrant pyroptosis may also be involved in the formation of a tumor-supportive microenvironment.
Therefore, gaining an in-depth understanding of the molecular pathways of pyroptosis and unraveling the complex connection between pyroptosis and cancer will help us make the most of pyroptosis and apply it to existing or new anticancer strategies.
Pyroptosis was first described in macrophages infected with S. typhurium and S. flexneri in the 1990s .
Although initially thought to be an apoptotic process, further studies have shown that this bacterial-induced cell death is critically dependent on caspase-1.
Pyroptotic cells share some features with apoptotic cells, such as chromatin condensation and DNA fragmentation, but can be distinguished by their intact nucleus, pore formation, cell swelling, and osmotic lysis.
Typically, pyroptotic cell rupture is achieved by pore-forming GSDM protein activation mediated by cysteine proteases after binding damage-associated molecular patterns ( DAMPs ) or pathogen-associated molecular patterns ( PAMPs ).
These same cysteine proteases may also directly or indirectly promote the maturation of pro-inflammatory cytokines that, together with DAMPs, initiate or sustain inflammatory responses when released.
Although pyroptosis plays an important protective role in pathogen breakdown, it has been implicated as a complicating factor in several human diseases, such as cardiovascular disease, neurodegenerative diseases, and AIDS.
Metabolic disorders such as diabetes may also promote pyroptosis through chronic inflammation and insulin-interfering cytokine production.
In cancer, the role of pyroptosis appears to be a double-edged sword. On the one hand, pyroptosis can rapidly lead to tumor regression, and on the other hand, it promotes the development of the tumor microenvironment.
Therefore, cancer cells may inhibit or stimulate pyroptosis to support their progression, depending on the environment.
Molecular mechanism of pyroptosis
Although the number of known pyroptotic pathways is likely to increase in the future, two main pathways and several alternative pathways have been elucidated.
Among the major pathways, pyroptosis is induced by GSDMD, involving inflammatory caspase-1 ( canonical pathway ) or caspase-4/5 ( non-canonical pathway ).
Among the alternative pathways, the one that has received the most attention is GSDME-induced pyroptosis via caspase-3.
Classical inflammasome pathway
In the canonical inflammasome pathway, pattern recognition receptors ( PRRs ) recognize DAMPs ( e.g. fibrinogen, heat shock proteins, DNA ) and/or PAMPs ( e.g. flagellin, glycans, lipopolysaccharides ) resulting in a process known as Activation of the corresponding cytoplasmic signaling complexes of the inflammasome, usually composed of sensor proteins, adapters, and effector caspases.
Although multiple PRRs, such as NLRs and TLRs, are involved in this process, only some of them are known to directly assemble the inflammasome and activate the cysteine protease caspase-1.
Specifically, PRRs/inflammasome sensors in this subset include NLRP1, NLRP3, NLRP4, AIM2, and pyrin of the NLR family.
Upon activation, most of these sensors interact with the adaptor protein ASC, which activates caspase-1 through the recruitment and cleavage of pro-caspase-1.
In addition to releasing and activating the lethal N-terminal domain of GSDMD ( GSDMD-N ), caspase-1 also matures pro-IL-1β and pro-IL-18 into IL-1β and IL-18, which pass through the necrotic membrane formed by GSDMD-N hole release.
non-classical inflammasome pathway
In contrast to the standard inflammasome pathway, the non-standard inflammasome pathway does not depend on caspase-1, but on caspase-4 and caspase-5 as well as caspase-11 in mice.
Activation of these caspases occurs through direct binding of LPS to the corresponding pre-cysteine proteases and bypasses inflammasome sensors.
Although these caspases do not directly activate IL-1β and IL-18, they trigger pyroptosis through GSDMD cleavage leading to potassium efflux, which activates the NLRP3 inflammasome and upregulates the action of caspase-1.
Studies have shown that in certain conditions, such as chemotherapy or targeted cancer therapy, an apoptosis-to-pyroptosis pathway can be induced through caspase-3.
Although caspases-3 is mainly involved in apoptosis execution and morphological changes, it can mediate pyroptosis by cleaving GSDME, which also leads to GSDME-N pore formation and membrane permeability changes.
When GSDME levels are high, caspase-3 activation rapidly triggers pyroptosis, but when GSDME levels are low, apoptosis is triggered. However, this concept still needs further verification.
In addition, there are several other alternative pyroptotic pathways, including cleavage of GSDMD by caspase-8; cleavage of GSDME by caspase-8 or granzyme B ( GzmB ) ; and cleavage of GSDMB by caspase-1 or granzyme A ( GzmA ) Cleavage of GSDMC by caspase-8, GSDMC through hypoxia-activated programmed death ligand 1 ( PD-L1 ) and pSTAT3 transcriptional upregulation and other unknown mechanisms to form GSDMA pores.
Pyroptosis and its components in cancer
The ambiguous role of pyroptosis in cancer appears to be related to cell type, genetics and duration of induction. Following aberrant expression and prolonged activity, GSDM, inflammasomes, and/or pro-inflammatory cytokines can promote tumors by inducing immunosuppressive cells, promoting epithelial-to-mesenchymal transition, and upregulating matrix metalloproteinases for extracellular matrix remodeling pathology.
On the other hand, in parallel with these effects, pyroptosis can also suppress tumors, for example, in a hepatocellular carcinoma model, pyroptosis induced by activation of the NLRP3 inflammasome significantly inhibited tumor metastasis and growth.
Given the dual role of pyroptosis, its molecular components are differentially expressed in different cancers.
GSDMs have been shown to simultaneously act as oncogenes or tumor suppressors to control proliferation, metastasis, therapy resistance, and antitumor immunity in tumors such as breast, gastric, cervical, and lung cancers.
In gastric cancer ( GC ), GSDMD expression was significantly reduced and resulted in enhanced tumor proliferation in vitro and in vivo.
Conversely, GSDMD protein levels are significantly elevated in non-small cell lung cancer ( NSCLC ) and are associated with tumor metastasis as well as worse prognosis.
Similar to GSDMD, the expression of GSDME was also decreased in gastric, breast and colorectal cancers.
The expression of GSDMC was significantly up-regulated in colorectal cancer. In colorectal cancer, GSDMC promoted carcinogenesis and proliferation in vitro and tumor growth in vivo.
The higher the GSDMB level, the higher the metastasis rate and the lower the survival rate of breast cancer patients.
Among other pyroptotic components, AIM2 expression was observed to be significantly reduced or absent in most colorectal cancer tumors and was associated with poor patient prognosis.
NLRP1 levels were also decreased in colorectal cancer tumor tissues and were associated with increased metastasis and decreased survival.
Caspase-1 mRNA levels were significantly reduced in breast cancer patient tissues, and caspase-1 deletion was associated with tumorigenesis in prostate and colorectal cancers.
Elucidating the relationship between pyroptosis and cancer requires more extensive research.
One challenge will be to identify the tumor-specific role of each pyroptotic molecular component.
Since multiple pathways contribute to pyroptosis and multiple components overlap, characterizing the overall tumor-specific effects of each pathway, rather than the individual effects of each component, may be useful in understanding and/or predicting tumor regulation of pyroptosis. more effective strategies.
The relationship between pyroptosis and antitumor immunity
The ability of cell death to trigger an adaptive immune response is called immunogenic cell death ( ICD ).
Unlike apoptosis, which is essentially a process of immune tolerance, pyroptosis has a molecular mechanism that induces a strong inflammatory response and is considered a form of ICD in some cases.
Although the link between pyroptosis and anticancer immunity is unclear, an increasing number of studies suggest that pyroptosis-mediated tumor clearance is achieved by enhancing immune activation and function.
We found that selective delivery of the mouse isoform Gsdma3 of GSDMA to human HeLa, mouse EMT6, and mouse 4T1 cancer cells resulted in pyroptosis in 20-40% of the cells.
In an in vivo model, three rounds of treatment by intravenous or intratumoral injection of NP-Gsdma3 resulted in significant tumor shrinkage.
Compared with PBS controls, NP–Gsdma3-treated tumors had increased numbers of CD4+, CD8+, natural killer ( NK ), and M1 macrophages, but monocytes, neutrophils, myeloid-derived suppressor cells, and M2 macrophages The number of cells decreases.
Furthermore, in addition to increased levels of IL-1β, IL-18 and HMGB1 at serum and tumor levels, up-regulation of many immune-stimulatory and anti-tumor effector genes ( such as Cd69, Gzma, Gzmb ), various immunosuppressive and tumor precursors were also found Genes ( eg Csf1, Vegfa, Cd274 ) are down-regulated.
By studying the expression of GSDM genes associated with CD8+ T cell markers by CTLs in lung squamous cell carcinoma ( LUSC ) and melanoma tumor samples, it was found that among the five GSDM gene members, only GSDMD expression correlated with CD8+ expression in CTLs T cell marker genes such as CD8A, CD8B, PRF1, GZMA, GZMB and IFNG were positively correlated. Further studies showed that GSDMD expression was significantly increased in activated CTLs of OT-1 mice compared with naive T lymphocytes.
Similarly, human CD8+ T cells upregulated GSDMD upon activation, and high levels of GSDMD protein were seen in tumor-infiltrating lymphocytes ( TILs ).
Furthermore, the cytotoxicity of CTL to 3LL-OVA cells was reduced after GSDMD knockout. Similar results were recorded using human CTL and the H1299 non-small cell lung cancer cell line.
Considering that a key pathway by which CTLs kill tumor cells is through the release of cytotoxic molecules into the immune synapses they form, it is speculated that entry of GSDMD and GzmB into effector cancer cells may be the underlying mechanism of CTL cytotoxicity identified in the study.
GSDMB-mediated pyroptosis is closely related to GzmA. Of the five human granzymes in HEK-293F cells, only GzmA was found to rapidly cleave GSDMB in a pattern similar to the NK cell killing assay.
When GzmA was electroporated into GSDMB-reconstituted 293T cells, it caused extensive GSDMB lysis and pyroptosis.
Likewise, GzmA-mediated cleavage of GSDMB is required for NK cell killing of 293 cells under physiological conditions.
Notably, other cancer cell lines with insignificant levels of GSDMB, such as OE33 ( esophageal cancer cells ) and HCC1954 ( breast cancer cells ), can be inhibited by exposure to cytokines normally released by activated CTLs such as IFN-γ and TNF . -α ) to transcriptionally induce increased GSDMB expression.
In turn, IFN-γ priming significantly enhanced pyroptosis in these cell lines, an effect ultimately dependent on GzmA.
Taken together, these findings not only suggest that GSDMB-mediated pyroptosis acts downstream of GzmA, but that cytotoxic lymphocytes can deliver GzmA to GSDMB-expressing cancer cells to promote antitumor immunity.
The study found that GSDME-mediated pyroptosis was associated with GzmB. Ectopic expression of mouse GSDME ( mGSDME ) in mouse 4T1E breast cancer cells significantly inhibited 4T1E tumor growth and resulted in increased infiltration of NK cells and tumor-associated macrophages ( TAMs ) in an in vivo model. Furthermore, GzmB- and perforin-expressing NK cells and CD8+ TILs increased in tumors when stimulated.
Further studies showed that the human NK cell line YT can activate pyroptosis in GSDME-expressing HeLa cells, and this induction is achieved by GzmB, which not only cleaves GSDME at the same location as caspase-3, but also indirectly activates caspase-3 .
Furthermore, CAR-T cells can induce GSDME-mediated pyroptosis of tumor cells in B-cell leukemia and solid tumor cells via perforin and GzmB release.
Treatment of human-derived macrophages with supernatants of co-cultured CD19-CAR-T cells and cancer cells ( NALM-6, Raji, or primary B leukemia cells ) promotes macrophage activation of caspase-1, cleavage of GSDMD, and release IL-6 and IL-1β.
We also found that ATP and HMGB1 in the co-culture supernatant were sufficient to promote macrophage IL-1β secretion and IL-6 upregulation, respectively.
Collectively, these findings suggest that CAR-T cell therapy induces CRS through GSDME-promoted pyroptosis, and analysis of tumor cells from patients before CD19-CART cell therapy showed that elevated GSDME levels were associated with more severe CRS.
Application prospect of pyroptosis in anticancer therapy
In recent years, an increasing number of studies have demonstrated the feasibility and therapeutic potential of using pyroptosis for antitumor immunity through different targeting and delivery approaches.
For example, methotrexate infused into cholangiocarcinoma ( CCA ) cells using tumor cell-derived microparticles ( TMPs ) to induce GSDME-mediated pyroptosis, which activates patient-derived macrophages and recruits neutrophils to Drug-directed tumor destruction at the tumor site.
Furthermore, when this methotrexate TMP delivery system was infused into the bile duct lumen of extrahepatic CCA patients, neutrophil activation and remission of biliary obstruction were observed in 25% of patients.
In addition, GSDME-mediated pyroptosis was also found to cause immune cell infiltration/activation in melanoma through a combination of BRAF and MEK inhibitors and lead to melanoma regression. In another strategy, metformin, the most commonly used drug for the treatment of type 2 diabetes, can inhibit cancer cell proliferation by indirectly activating pyroptosis through caspase-3.
A series of small-molecule inhibitors targeting KRAS, EGFR, or ALK mutant lung cancers have also been found to induce pyroptosis through caspase-3-mediated GSDME cleavage after activation of the intrinsic mitochondrial apoptotic pathway.
In breast cancer cells, treatment with RIG-1 agonists triggers extrinsic apoptotic pathways and pyroptosis, activates STAT1 and NF-κB and upregulates lymphocyte recruitment of chemokines.
Thus, following RIG-1 activation in mice, a reduction in breast cancer metastasis and tumor growth was accompanied by an increase in tumor lymphocytes.
Another major hurdle facing almost all anticancer immunotherapy strategies is the dysregulation of the immunosuppressive tumor microenvironment. To address this issue, Lu et al. engineered NK92 cells containing a chimeric costimulatory transforming receptor ( CCCR ), which converts inhibitory PD-1 signals into activating signals, effectively enhancing antitumor activity. In vitro, CCCR-NK92 cells rapidly killed H1299 cells via GSDME-mediated pyroptosis and significantly inhibited tumor growth in vivo.
In addition, more and more exciting research reports show that pyroptosis induction synergizes with PD-1 inhibitors to turn tumors from “cold” to “hot”, indicating the great potential of this combination.
As an inflammatory cell death mode, pyroptosis plays an important role in tumor suppression by stimulating anti-tumor immune responses. In some cases, induction of pyroptosis alone may be sufficient to retard tumor growth.
However, one of the greatest challenges for the therapeutic application of pyroptosis is the irregularity in the expression and function of pyroptosis-related components, not only between different cancers, but also within the same type of cancer.
Nonetheless, advances in molecular, genetic, and epigenetic targeting/delivery systems, as well as the development of precise and personalized medicine, give hope that we will soon be able to harness these powerful mechanisms as tools for the treatment of cancer.
1. Pyroptosis at the forefront of anticancerimmunity. J Exp Clin Cancer Res. 2021; 40: 264.
Understand Tumor Immunity and Pyroptosis to find the anti-cancer strategy
(source:internet, reference only)