Ovarian cancer (OC) is the most deadliest gynecological malignancy (Siegel et al., 2016; Matulonis et al., 2016). High-grade serous ovarian cancer (HGSOC) is the most common and lethal form of OC (Torre et al., 2018; Berkel and Cacan, 2021). The current standard of care includes cytoreductive surgery and primary chemotherapy (Gadducci et al., 2019). OC patients are also poorly responsive to new immunotherapy applications; therefore, these treatment options still remain insufficient to confer long-term survival benefit.
Pyroptosis is a lytic form of programmed cell death with a highly pro-inflammatory nature. Gasdermins (GSDMs) are members of a family of pore-forming effector proteins, leading to pyroptotic cell death upon activation by certain stimuli. These proteins, excluding PJVK, lead to membrane permeabilization (Kayagaki et al., 2015; Shi et al., 2015; He et al., 2015). The formation of pores in the cell membrane by gasdermins allow the release of multiple proinflammatory molecules, including mature IL-1β and IL-18 as well as small DAMPs (Broz et al., 2020). These N- and C-terminal domains of gasdermins are connected by a central flexible linker (except for PJVK) which be cleaved by certain caspases upon induction by pathogen-derived or host-derived danger signals. Following the release of intramolecular inhibition on N-terminal domain due to proteolytic cleavage of linker region, this fragment localizes into cell membranes to form large oligomeric pores. The ultimate release of cytoplasmic contents from pyroptotic cells provides signals enough to initiate an inflammatory cascade.
Different caspases are responsible for the specific cleavage of certain gasdermins within their central flexible linker regions. These inflammatory caspases are activated by various inflammasomes upon induction by different stimuli (Ding et al., 2016; Demarco et al., 2020; Chen et al., 2019; Orning et al., 2018; Sarhan et al., 2018; Sanjo et al., 2019). Likewise, these caspases can only cleave specific gasdermin proteins at certain regions to activate them. For example, GSDMD can be cleaved at a certain amino acid position by inflammatory caspases including caspase-1, -4, -5, -11 or by caspase-8. Similarly, caspase-3 and − 8 have been identified as proteases responsible for the cleavage of GSDME and GSDMC, respectively (Wang et al., 2017; Rogers et al. 2017; Hou et al., 2020).
Gasdermin protein family in humans currently has six paralogous members: GSDMA, GSDMB, GSDMC, GSDMD, GSDME and PJVK (Broz et al., 2020). The expression of GSDMA was shown to be negatively regulated in gastric cancer (Saeki et al., 2000). GSDMB was found to be involved in tumor progression in multiple cancer types (Carl-McGrath et al., 2008; Sun et al., 2008; Hergueta-Redondo et al., 2014). Another study reported that the expression of GSDMC is upregulated in metastatic melanoma cells (Watabe et al., 2001). Others suggested that GSDMC, GSDMD and GSDMA may be potential tumor suppressors and GSDMB, which was amplified and upregulated in some gastric cancers, could function as an oncogene (Saeki et al., 2009). Contrarily, GSDMC was reported to contribute to tumorigenesis in colorectal cancer, since its knockdown led to decreased proliferation in colorectal cancer cell lines (Miguchi et al., 2016). Zhang et al. found that GSDME limits tumor growth by promoting pyroptosis (2020). Authors showed that pyroptosis activates anti-tumor immunity through the augmentation of the tumor cell phagocytosis by tumor-associated macrophages and also by boosting the number and activity of tumor-infiltrating NK and CD8+ T lymphocytes (Zhang et al., 2020). Similar to GSDME, GSDMB was also found to increase anti-tumor immunity (Zhou et al., 2020). Authors showed that NK- or cytotoxic T lymphocyte-derived granzyme A cleaves GSDMB in GSDMB-positive cells to promote its pore-forming activity, leading to pyroptotic killing of target cancer cells (Zhou et al., 2020).
Recently, we found that GSDMD and GSDMC show increased expression, and in contrast, GSDME and PJVK show decreased expression in serous OC compared to normal ovaries, at the mRNA level (Berkel and Cacan, 2021). We also showed that the percentage of copy number gain events in genes encoding GSDMC and GSDMD is around 50% in OC patients, in parallel to their increased expression. Moreover, the percentage of CNV events in genes encoding GSDMC, GSDMD, GSDME and PJVK is the highest in OC among all cancer types. In contrast to other gasdermins, GSDME are mostly characterized by copy number losses in OC, again in line with its decreased expression in this cancer type.
A recent report identified cell-surface protein NINJ1 as an important mediator of plasma membrane rupture in lytic cell death pathways including pyroptosis, and showed that NINJ1 is an essential protein for pyroptosis-related plasma membrane rupture (Kayagaki et al., 2021; Wang and Shao, 2021). Authors showed that NINJ1−/− cells are able to release small molecules such as IL-1β through gasdermin pores; however, NINJ1 is required for the release of larger molecules that propagate inflammation.
In the present study, we showed GSDMD protein levels are increased in ovarian tumor compared to adjacent non-malignant stromal cells. In contrast, GSDME protein levels are decreased in ovarian tumor relative to surrounding normal stromal cells. We also found that NINJ1 expression is decreased in late stage serous OC compared to early stage, and that NINJ1 copy number loss events is the highest in OC among other cancer types. Moreover, we reported that low NINJ1 expression is associated with worse overall survival. In addition, we observed that cisplatin-resistant OC cell lines have lower NINJ1 levels compared to cisplatin-sensitive cells, again suggesting that decreased NINJ1 levels might contribute poorer disease outcomes in OC. This study points that NINJ1 might be an important player in ovarian cancer ininitiation, progression and therapy response.