Nerve injury-induced protein 2 preserves lysosomal membrane integrity to suppress ferroptosis

eLife Assessment

This fundamental work uncovers an unexpected lysosomal function for NINJ2 and links it to ferroptosis and cancer biology. The evidence supporting the conclusions appears to be convincing. This work will be of general interest to the community of ferroptosis and cancer biology.

https://doi.org/10.7554/eLife.110919.3.sa0

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Abstract

Nerve injury-induced protein 1 (NINJ1), a cell adhesion molecule, is oligomerized during lytic cell death and mediates plasma membrane rupture to release large intracellular molecules that propagate the inflammatory response. We and others previously showed that NINJ2, a close relative of NINJ1, does not promote plasma membrane rupture to spread inflammation. Here, we identify that NINJ2 is necessary for lysosome membrane integrity to protect cells from ferroptosis. Specifically, we found that NINJ2 localizes to lysosomes and interacts with LAMP1, an anchor glycoprotein of the lysosome membranes and a sensor of stressed lysosomes. We also found that loss of NINJ2 exacerbates lysosomal membrane permeabilization (LMP), which allows for selective leakage of lysosomal contents, such as labile iron, into the cytosol. Accordingly, loss of NINJ2 elevates cellular labile iron accumulation and decreases expression of ferritins, the primary intracellular iron storage protein complexes. Mechanistically, we found that loss of NINJ2 promotes ferritin FTH degradation in lysosomes, which can be reversed by knockdown of LAMP1. Moreover, we found that loss of NINJ2 sensitizes cells to ferroptosis induced by RSL3 and Erastin, consistent with a recent study that loss of NINJ2 predisposes mice to chronic inflammation. Together, these findings uncover a previously unrecognized activity of NINJ2 from lysosome homeostasis to ferroptosis, which can be explored as a cancer therapeutic strategy, especially considering that NINJ2 and ferritins are found to be overexpressed and positively associated with iron-addicted cancers.

Introduction

Ninjurin 2 (Nerve injury-induced protein 2; NINJ2), along with NINJ1, belongs to the Ninjurin family of homophilic cell-surface adhesion molecules (Araki and Milbrandt, 2000; Araki and Milbrandt, 1996). These proteins were originally identified as being upregulated in Schwann cells and dorsal root ganglion neurons following peripheral nerve injury. Subsequent studies showed that NINJ1 and NINJ2 promote neurite outgrowth and facilitate interactions between Schwann cells and regenerating axons, thereby contributing to effective nerve repair (Araki and Milbrandt, 2000; Araki and Milbrandt, 1996; Tomita et al., 2019). Structural analysis indicated that NINJ1 and NINJ2 share approximately 52–55% amino acid sequence identity and 67% sequence similarity. They are two-pass transmembrane proteins composed of an extracellular N-terminal domain, two conserved hydrophobic transmembrane domains, and a C-terminal extracellular domain (Araki et al., 1997). Despite high sequence homology, recent studies showed that NINJ1 and NINJ2 exert divergent functions potentially due to their structural variations. For instance, NINJ1 mediates plasma membrane rupture by polymerizing into straight, amphipathic filaments that disrupt membrane integrity (Kayagaki et al., 2021; Mossemann, 2023; Sahoo et al., 2025). In contrast, NINJ2 assembles into curved filaments that are unable to permeabilize the membrane and do not promote plasma membrane rupture (Sahoo et al., 2025). These data indicate that NINJ1 and NINJ2 exhibit both overlapping and distinct functions across diverse biological processes.

Recent studies have shown that NINJ2 is a multifaceted protein with profound biological functions. NINJ2 is found to be highly expressed in immune-related tissues, including bone marrow, lymph nodes, spleen, and thymus (Liu et al., 2024; Zhang et al., 2024b), suggesting a role in immune regulation. Indeed, studies have shown that NINJ2 acts as a pro-inflammatory mediator by physically interacting with TLR4, activating the NF-κB pathway to promote the expression of inflammatory markers like IL-6 and TNF-α (Wang et al., 2017; Liu et al., 2025; Peroni and Sorosina, 2018). Additionally, we found that loss of NINJ2 leads to activation of NLRP3 inflammasome and subsequently promotes pyroptosis, a pro-inflammatory programmed cell death that leads to the dysregulated release of cytokines like IL-1β and IL-18 (Zhang et al., 2024b). Furthermore, we recently identified NINJ2 as a transcriptional target of the tumor suppressor p53 and that NINJ2, in turn, represses p53 mRNA translation, implicating its role in tumorigenesis (Zhang et al., 2024a). Finally, NINJ2 gene polymorphisms are found to be associated with increased risk of ischemic stroke, coronary artery disease, multiple sclerosis (MS), and Alzheimer’s disease (Wan et al., 2011; Noroozi et al., 2019; Cheng et al., 2021; Wang et al., 2021), suggesting a role in vascular and neuroinflammatory pathologies. Together, these data indicate that NINJ2 participates in a wide range of physiological and pathological processes, yet the molecular mechanisms underlying its multifunctional roles remain poorly understood.

Lysosomes are central regulators of cellular homeostasis, coordinating protein degradation, autophagy, and metabolic signaling (Lamming and Bar-Peled, 2019). Lysosomal membrane permeabilization (LMP) is defined by the formation of ultrastructurally undetectable, tiny pores at the lysosomal membrane and thereby causes selective release of lysosomal contents into the cytosol (Wang et al., 2018; Serrano-Puebla and Boya, 2016). Recently, it was found that low-grade LMP does not always cause cell death but can be repaired by the ESCRT complex (Radulovic et al., 2018). It was also found that LMP triggers the activation of NLRP3 inflammasome and subsequently elicits an immune response (Katsnelson et al., 2016; Heid et al., 2013). We previously reported that NINJ2 deficiency leads to NLRP3 inflammasome activation (Zhang et al., 2024b). However, it was not certain whether this activation was linked to lysosomal damage. To address this, we examined the role of NINJ2 in regulating lysosomal morphology and function. We found that NINJ2 localizes to lysosomes and loss of NINJ2 exacerbates LMP. We also found that the LMP mediated by NINJ2-deficiency results in increased levels of labile iron along with decreased expression of ferritin. We further demonstrated that NINJ2-deficiency promotes degradation of ferritin and subsequently enhances ferroptosis, a regulated form of cell death driven by iron-dependent lipid peroxidation. Together, these findings reveal a previously unrecognized function of NINJ2 in linking lysosomal homeostasis to ferroptosis.

Results

Ninj2 protein localizes to lysosomes and interacts with LAMP1

To determine whether NINJ2 is involved in modulating lysosomal activity, we first examined whether NINJ2 localizes to lysosomes. To this end, MCF7 cells expressing Flag-tagged NINJ2 were stained with LysoTracker to label lysosomes and with an anti-Flag antibody to detect NINJ2. Confocal microscopy revealed substantial co-localization of NINJ2 with LysoTracker-positive vesicles, indicating that some NINJ2 proteins localize to lysosomes (Figure 1A). To further test this, MCF7 cells expressing Flag-tagged NINJ2 were co-stained with antibodies against NINJ2 and lysosome-associated membrane protein 1 (LAMP1), a well-established lysosomal marker (Eskelinen, 2006; Carlsson and Fukuda, 1989; Carlsson et al., 1988). Consistent with the data from LysoTracker staining (Figure 1A), we found that NINJ2 was co-localized with LAMP1 in lysosomes (Figure 1B), confirming that NINJ2 localizes to lysosomes. Next, we examined whether NINJ2 physically interacts with LAMP1 by performing reciprocal immunoprecipitation assays using 293T cells expressing Flag-tagged NINJ2. We found that endogenous LAMP1 was detectable in NINJ2-immunocomplex when immunoprecipitated with an anti-Flag antibody (Figure 1C). Conversely, Flag-tagged NINJ2 was readily detected in LAMP1-immunocomplexes when immunoprecipitated with an anti-LAMP1 antibody (Figure 1D). To verify that NINJ2 interacts with LAMP1, we performed a proximity ligation assay (PLA) (Söderberg et al., 2006), which detects protein-protein interactions at a subcellular level. Briefly, MCF7 cells expressing Flag-tagged NINJ2 were incubated with anti-Flag and/or anti-LAMP1 antibodies. PLA signals were visualized as discrete fluorescent puncta. We observed robust PLA signals in cells stained with both antibodies, indicating close interaction between NINJ2 and LAMP1 (Figure 1E). In contrast, no PLA signals were detected in control conditions in which cells were incubated without any primary antibody or with only one antibody (Figure 1E).

Figure 1

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Nerve injury-induced protein 2 (NINJ2) protein localizes to lysosomes and interacts with lysosome-associated membrane protein 1 (LAMP1).

(A) MCF7 cells were transiently transfected with a plasmid expressing Flag-tagged NINJ2, followed by immunostaining with DAPI, LysoDye and Flag. The arrow indicates co-staining of NINJ2 and LysoDye. (B) MCF7 cells were transiently transfected with a plasmid expressing Flag-tagged NINJ2, followed by immunostaining with anti-Flag and anti-LAMP1. (**C–D**) 293T cells were transiently transfected with Flag-tagged NINJ2 plasmid for 24 hr, followed by immunoprecipitation with IgG, anti-Flag (C) or anti-LAMP1 (D). The immunocomplex was examined by Western blot analysis with Flag or LAMP1 antibody. (E) MCF7 cells were transfected with Flag-tagged NINJ2 antibody, followed by proximity ligation assay (PLA) assay as described in ‘Materials and methods.’ The positive PLA signal is shown in red puncta.

Figure 1—source data 1 Original gel images with labels.: https://cdn.elifesciences.org/articles/110919/elife-110919-fig1-data1-v1.zip
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Loss of NINJ2 leads to enhanced lysosomal membrane permeability along with increased expression of LAMP1

The localization of NINJ2 in lysosomes prompted us to determine whether NINJ2 regulates lysosomal function. To this end, isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with L-leucyl-L-leucine methyl ester (LLOMe), a well-established inducer of lysosomal membrane permeabilization (LMP) (Thiele and Lipsky, 1992; Thiele and Lipsky, 1990), followed by immunostaining with Galectin-3 and LAMP1. We would like to note that in response to lysosomal damage, Galectin-3 rapidly translocalizes to lysosomal membranes where it participates in lysosomal repair and removal (Aits et al., 2015; Jia et al., 2020). Thus, colocalization of Galectin-3 with LAMP1 serves as a key indicator of LMP. We found that in the absence of LLOMe, little colocalization of Galectin-3 and LAMP1 was observed in isogenic control and NINJ2-KO MCF7 cells (Figure 2A, top two panels), indicating minimal lysosomal membrane damage (Figure 2A, compare top two ‘MERGE’ panels). Upon treatment with LLOMe, isogenic control cells exhibited moderate co-staining of Galectin-3 and LAMP1 (Figure 2A, Iso. Ctrl + LLOMe panel). Strikingly, the recruitment of Galectin-3 to LAMP1-positive lysosomes was markedly enhanced in NINJ2-KO MCF7 cells, suggesting that NINJ2 deficiency exacerbates LLOMe-induced lysosomal membrane permeabilization (Figure 2A, NINJ2-KO+LLOMe panel). In addition to elevated LMP mediated by NINJ2-deficiency, we observed that LAMP1 staining was enhanced in NINJ2-KO MCF7 cells compared to isogenic controls regardless of LLOMe treatment (Figure 2A, LAMP1 panels), suggesting that NINJ2 deficiency alters LAMP1 expression. To verify this, isogenic control and NINJ2-KO Molt4 cells were mock-treated or treated with LLOMe or glucose oxidase (GO), another LMP inducer (Eriksson et al., 2023), followed by measurement of LAMP1 expression. We found that loss of NINJ2 resulted in a marked increase in LAMP1 expression regardless of LLOMe or GO treatment (Figure 2B–C, LAMP1 panel, compare lanes 1, 3, and 5 with 2, 4, and 6, respectively). In contrast, LLOMe and GO did not significantly alter LAMP1 expression in these cells. Similarly, loss of NINJ2 led to increased LAMP1 expression in MCF7 cells regardless of LLOMe or GO treatment (Figure 2D–E). Consistent with this, we also showed that the level of LAMP1 transcripts was increased by loss of NINJ2 regardless of LLOME treatment in both Molt4 and MCF7 cells (Figure 2—figure supplement 1). Together, these data suggest that loss of NINJ2 leads to low-grade lysosomal membrane damage accompanied by elevated LAMP1 expression, which in turn exacerbates LMP.

Figure 2 with 1 supplement see all

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Loss of Nerve injury-induced protein 2 (NINJ2) leads to enhanced lysosome-associated membrane protein 1 (LAMP1) expression and lysosomal membrane permeability.

(A) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with L-leucyl-L-leucine methyl ester (LLOMe) (1 μM) for 5 hr, followed by immunostaining with antibodies against Galectin 3 and LAMP1. Scale bar: 20 μM. (**B–C**) Isogenic control and NINJ2-KO Molt4 cells were mock-treated or treated with LLOMe (B) or glucose oxidase (GO) (C) for 5 hr. The cell lysates were subjected to Western blot analysis using antibodies against NINJ2, LAMP1, and actin. The relative protein level of LAMP1 in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane. (**D–E**) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with LLOMe (D) or GO (E) for 5 hr, followed by Western blot analysis to detect expression of LAMP1 and actin. The relative protein level of LAMP1 in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane.

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Loss of NINJ2 elevates intracellular labile iron level and inhibits ferritin expression

LMP is known to release lysosomal contents, such as redox-active Fe²⁺, from lysosomes into the cytosol, thereby expanding the labile iron pool and promoting lipid peroxidation (Saimoto et al., 2025). Thus, we measured the level of labile iron levels in isogenic control and NINJ2-KO Molt4 and MCF7 cells treated with Ferric Ammonium Citrate (FAC), an iron source. We found that the level of intracellular labile iron was markedly increased by loss of NINJ2 in both Molt4 and MCF7 cells (Figure 3A–B). To verify that NINJ2-deficiency alters iron homeostasis, we measured the expression of ferritin heavy chain (FTH) and ferritin light chain (FTL) in isogenic control and NINJ2-KO Molt4 cells treated with or without FAC. FTH and FTL are oligomerized to form ferritins, the primary intracellular iron storage protein complexes (Kotla et al., 2022; Melino et al., 1978). FTH exerts ferroxidase activity by catalyzing Fe²⁺ to Fe³⁺, whereas FTL helps promote iron nucleation and mineralization for long-term storage. In response to FAC treatment, both FTL and FTH proteins were elevated as expected (Figure 3C–D, FTL and FTH panels, compare lane 1 with 3). Importantly, loss of NINJ2 markedly decreased expression of FTL and FTH (Figure 3C–D, compare lane 1 and 3 with lane 2 and 4, respectively), suggesting that NINJ2 is required for FTH and FTL expression. Additionally, we found that loss of NINJ2 markedly reduced FTH and FTL expression in MCF7 cells regardless of FAC treatment (Figure 3E–F, compare lane 1 and 3 with lane 2 and 4, respectively).

Figure 3

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Loss of Nerve injury-induced protein 2 (NINJ2) leads to increased level of labile iron and reduced expression of ferritin.

(**A–B**) Isogenic control and NINJ2-KO Molt4 (A) and MCF7 (B) cells were treated with Ferric Ammonium Citrate (FAC) (30 mg/ml) for 24 hr. Cell lysates were collected and the level of labile iron was measured using the QuantiChrom assay kit. * Indicated p<0.05 by Student’s t-test. (**C–D**) Isogenic control and NINJ2-KO Molt4 cells were mock-treated or treated with FAC (30 mg/ml) for 16 hr, followed by Western blot analysis to measure the level of NINJ2 (C), ferritin light chain (FTL) (C), ferritin heavy chain (FTH) (D), and actin (**C–D**). The relative protein levels of FTL (C) and FTH (D) in control cells were arbitrarily set as 1.0 and the relative fold change was shown below each lane. (**E–F**) Isogenic control and NINJ2-KO MCF7 cells were mock-treated or treated with FAC (30 mg/ml) for 16 hr, followed by Western blot analysis to measure the level of FTL (E), FTH (F), and actin (**E–F**). The relative protein levels of FTL (E) and FTH (F) in control cells were arbitrarily set as 1.0 and the relative fold change was shown below each lane.

Figure 3—source data 1 Original gels with labeling.: https://cdn.elifesciences.org/articles/110919/elife-110919-fig3-data1-v1.zip
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Loss of NINJ2 promotes ferritin degradation

To uncover the mechanism by which NINJ2 regulates ferritin expression, we first examined the level of FTH transcripts and found it not to be altered by loss of NINJ2 (Figure 4A). Next, the half-life of FTH protein was measured in isogenic control and NINJ2-KO MCF7 cells treated with cycloheximide for various times. We found that the half-life of FTH protein decreased from 23.1 hr in isogenic control cells to 11.95 hr in NINJ2-KO cells (Figure 4B–C), indicating that NINJ2 is required for maintaining FTH protein stability. Since FTH is known to be degraded primarily in lysosomes via NCOA4 (Radisky and Kaplan, 1998; Asano et al., 2011), we then asked whether LAMP1 plays a role in the decrease in FTH expression by loss of NINJ2. To this end, two siRNAs against LAMP1 were designed and transiently transfected into isogenic control and NINJ2-KO MCF7 cells. As expected, the level of LAMP1 protein was diminished upon siRNA transfection (Figure 4D, LAMP1). Importantly, knockdown of LAMP1 led to increased expression of FTH and FTL in both isogenic control and NINJ2-KO MCF7 cells (Figure 4D). Together, these data indicated that loss of NINJ2 promotes ferritin turnover in lysosomes, which can be reversed by knockdown of LAMP1.

Figure 4

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Loss of Nerve injury-induced protein 2 (NINJ2) promotes ferritin degradation.

(A) The level of ferritin heavy chain (FTH) and HPRT transcripts was measured in isogenic control and NINJ2-KO Molt4 cells. The relative level of FTH transcripts in control cells was arbitrarily set as 1.0 and the relative fold change of transcripts was shown below each lane. (B) Isogenic control and NINJ2-KO MCF7 cells were treated with cycloheximide (50 μg/mL) for 3 to 15 hr. The cell lysates were collected and subjected to Western blot analysis using FTH and actin antibodies. (C) The levels of FTH and actin protein in (B) were quantified and the relative protein half-life of FTH was calculated using GraphPad Prism software. (D) Isogenic control and NINJ2-KO MCF7 cells were transiently transfected with scrambled siRNA or siRNAs against LAMP1 for 3 days, followed by Western blot analysis with antibodies against lysosome-associated membrane protein 1 (LAMP1), FTH, ferritin light chain (FTL), and actin. The relative level of LAMP1, FTH, and FTL protein in control cells was arbitrarily set as 1.0 and the relative fold change of transcripts was shown below each lane.

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Loss of NINJ2 promotes ferroptosis

Recent studies have shown that LMP releases reactive iron into the cytosol and promotes lipid peroxidation, thereby serving as a critical initiating event in ferroptosis, an iron-dependent form of programmed cell death (Saimoto et al., 2025; Cañeque et al., 2025). Since loss of NINJ2 increases the level of labile iron (Figure 3A–B), we thus examined whether NINJ2 modulates ferroptosis. As FTH serves as a protector against ferroptosis, its expression was first examined in isogenic control and NINJ2-KO cells treated with or without RSL3 and Erastin, both of which are ferroptosis inducers (Dixon et al., 2012; Yang and Stockwell, 2008; Yang et al., 2014). Indeed, we found that the level of FTH was decreased by NINJ2-KO regardless of RSL3 or Erastin treatment in both Molt4 and MCF7 cells (Figure 5A–B), suggesting that NINJ2-deficiency promotes ferroptosis. To further test this, isogenic control and NINJ2-KO Molt4 cells were treated with various doses of RSL3 or Erastin, and cell viability was assessed. We found that loss of NINJ2 significantly sensitized Molt4 cells to RSL3 treatment, reducing the IC₅₀ from 0.31 μM in isogenic control cells to 0.16 μM in NINJ2-KO cells (Figure 5C). Similarly, NINJ2 deficiency enhanced the sensitivity of Molt4 cells to Erastin, as evidenced by decreased IC_₅₀ in NINJ2-KO cells (Figure 5D). To validate these findings, isogenic control and NINJ2-KO MCF7 cells were treated with RSL3 or Erastin. Consistently, we found that loss of NINJ2 promoted ferroptosis in MCF7 cells, as indicated by a marked reduction in their IC₅₀ values (Figure 5E–F). To further verify this, a colony formation assay was performed. We found that under mock treatment conditions, NINJ2-KO suppressed colony formation, consistent with previous reports (Zhang et al., 2024a). Importantly, RSL3 and Erastin markedly reduced colony formation in MCF7 cells, which was further inhibited by NINJ2-KO (Figure 5G–H).

Figure 5

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Loss of Nerve injury-induced protein 2 (NINJ2) promotes ferroptosis.

(A) Isogenic control and NINJ2-KO Molt4 cells were treated with RSL3 (1.25 μM) or Erastin (2.5 μM) for 8 hr, and the levels of NINJ2, ferritin heavy chain (FTH), and actin were measured by Western blot analysis. The relative protein level of FTH in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane. (B) Isogenic control and NINJ2-KO MCF7 cells were treated with RSL3 (1.25 μM) for 8 hr, followed by Western blot analysis to detect FTH and actin. The relative protein level of FTH in control cells was arbitrarily set as 1.0 and the relative fold change was shown below each lane. (**C–D**) Isogenic control and NINJ2-KO Molt4 cells were treated with RSL3 (C) or Erastin (D) from 0 to 7.29 μM for 48 hr. The relative cell viability was measured using the CellTiter-Glo Viability Assay kit. The relative cell viability is calculated as a percentage of untreated (control) cells. Data points represent the mean ± SD from four representative experiments. Curves were fitted using nonlinear regression in GraphPad Prism Software. IC₅₀ values represent the drug concentration required to achieve 50% inhibition of maximal proliferation capacity. (**E–F**) Isogenic control and NINJ2-KO MCF7 cells were treated with RSL3 (E) or Erastin (F) from 0 to 7.29 μM for 48 hr, followed by cell viability assay. The IC₅₀ was calculated with nonlinear regression analysis using GraphPad Prism Software. (**G–H**) Colony formation was performed with isogenic control and NINJ2-KO MCF7 cells were treated with or without RSL3 or Erastin for 24 hr. The drugs were then withdrawn to allow colonies to grow for 3 weeks.

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NINJ2, FTH1, and FTL are overexpressed in hepatocellular and breast carcinomas and are positively associated among one another

Previous reports have shown that ferritins promote tumorigenesis through regulating iron homeostasis and oxidative stress, as well as suppressing ferroptosis. To determine whether the NINJ2-ferritins axis contributes to cancer development, we searched the TCGA database for the association between NINJ2 and ferritin in several types of human cancers. Interestingly, we found that NINJ2, FTH, and FTL were all upregulated in hepatocellular carcinoma (Figure 6A–C) and breast cancer (Figure 6D–F), both of which are iron-addicted malignancies. Furthermore, we found that NINJ2 expression was positively associated with FTH and FTL in both hepatocellular carcinoma (Figure 6G–H) and breast carcinoma (Figure 6I–J). Together, these findings suggest that NINJ2 may function in concert with ferritin to promote tumor cell growth especially in iron-addicted tumors, which may represent a therapeutic vulnerability for these types of cancers.

Figure 6

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Nerve injury-induced protein 2 (NINJ2) and ferritins are overexpressed in hepatocellular and breast carcinomas and are positively associated with one another.

(**A–C**) Boxplot shows the relative expression of NINJ2 (A), ferritin heavy chain (FTH) (B), and ferritin light chain (FTL) (C) in normal and hepatocellular carcinomas. The analysis was performed using UACLAN database. (**D–F**) Boxplot shows the relative expression of NINJ2 (A), FTH (B), and FTL (C) in normal and breast carcinomas. The analysis was performed using UACLAN database. (G) NINJ2 expression is positively associated with FTH (G) in hepatocellular carcinomas. The analysis was performed using the GEPIA2 database (http://gepia2.cancer-pku.cn/#correlation). Statistical analysis suggests a strong correlation between NINJ2 and FTH expression in hepatocellular carcinomas (Pearson’s r=0.54). (H) NINJ2 expression is positively associated with FTL (H) in hepatocellular carcinomas (Pearson’s r=0.54). (**I–J**) NINJ2 expression is positively associated with FTH (I, Pearson’s r=0.54) and FTL (J, Pearson’s r=0.67) in breast carcinomas.

Discussion

NINJ2 is a multifaceted protein and participates in various biological and pathological processes. However, the underlying mechanisms underlying these processes are not fully understood. Here, we identify NINJ2 as a critical regulator of lysosomal integrity and ferroptosis. Specifically, we demonstrate that NINJ2 localizes to lysosomes and interacts with LAMP1, and that loss of NINJ2 enhances LMP. We also found that loss of NINJ2 increases the level of cytosolic labile iron by promoting lysosomal ferritin degradation. Consequently, NINJ2 deficiency sensitizes cells to ferroptosis. Finally, we showed that NINJ2 and ferritin are co-overexpressed and positively correlated in several iron-addicted cancers, including hepatocellular carcinoma and breast cancer, suggesting that the NINJ2-FTH axis may be targeted for these types of cancer. These data let us speculate that loss of NINJ2 leads to low-grade lysosomal membrane damage, leading to leakage of labile iron into the cytosol. The released iron increases oxidative stress and promotes ferritin degradation in lysosomes, which further elevates cytosolic iron levels, creating a feed-forward loop that sensitizes cells to ferroptosis. A model to elucidate the role of NINJ2 in maintaining lysosomal membrane integrity and iron homeostasis is proposed in Figure 7.

Figure 7

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A model to elucidate the role of Nerve injury-induced protein 2 (NINJ2) in maintaining lysosomal membrane integrity and iron homeostasis.

Another interesting observation is that NINJ2 is required for Ferritin expression. Our data indicated the NINJ2-deficiency leads to reduced expression of Ferritin (Figure 3C–F). Moreover, we found that reduced Ferritin expression by loss of NINJ2 is due to enhanced lysosomal degradation (Figure 4B–C). In support of this, we showed that knockdown of LAMP1 reverses the inhibition of ferritin expression observed in NINJ2-knockout cells (Figure 4D). Although not yet experimentally confirmed, we tentatively propose that the enhanced degradation of ferritin in NINJ2-KO cells results from increased labile iron in these cells owing to the low-grade LMP. On the other hand, the NINJ2-Ferritin axis may be explored as viable strategy for cancer management. In support of this notion, we found that NINJ2 is positively associated with ferritins in iron-addicted cancers, such as HCC and breast cancer (Figure 6). Notably, most iron-addicted cancers rely on intact lysosomal function to shield excess iron and evade oxidative stress. Thus, targeting NINJ2 may unmask a convergent vulnerability, rendering tumor cells more susceptible to ferroptosis. Notably, we have developed a small peptide derived from the N-terminal extracellular domain that can enhance pyroptosis (Zhang et al., 2025). It would be interesting to determine whether this peptide can enhance ferroptosis.

Recent studies have shown that lysosomal leakage does not always lead to cell death since minor lysosomal membrane damage can be repaired via the ESCRT complexes (Radulovic et al., 2018; Skowyra et al., 2018). Interestingly, low-grade lysosomal leakage can lead to limited release of lysosomal contents, such as iron and acid hydrolases, thereby influencing diverse cellular processes, such as inflammatory responses (Stahl-Meyer et al., 2021). The finding that loss of NINJ2 leads to low-grade LMP (Figure 2) is consistent with previous observations that loss of NINJ2 promotes pyroptosis and inflammation (Zhang et al., 2024b). How does loss of NINJ2 lead to LMP? One possibility is that loss of NINJ2 may alter membrane fluidity through lipid remodeling. Indeed, we showed previously that loss of NINJ2 alters lipid metabolism and NINJ2 deficiency leads to a marked increase in ceramide levels (Zhang et al., 2024b). Notably, ceramide is a critical lipid mediator that destabilizes lysosomal membranes, acting as a potent trigger for LMP and subsequent cell death. Another possibility is through forming a complex with LAMP1 (Figure 1B–E). We postulate that the NINJ2-LAMP1 complex acts as a protective scaffold on the lysosome surface and thereby prevents the leakage of lysosomal content. Interestingly, we also observed that NINJ2-KO leads to increased LAMP1 expression at both transcript and protein levels (Figure 2B-E, Figure 2—figure supplement 1), which may be a compensatory protective response. Indeed, elevated LAMP1 levels have been found to stabilize damaged lysosomal membranes and facilitate lysosomal repair, thereby preventing cells from death induced by membrane rupture (Mindell, 2012). LAMP1 transcript is known to be upregulated by TFEB (Transcription Factor EB), a master regulator of the lysosomal-autophagy system (Medina et al., 2011). When LMP occurs, TFEB can translocate to nucleus and activate several lysosome-related genes, including LAMP1. Notably, phosphorylation of TFEB by mTORC1 at Serine 211 will inactivate TFEB by preventing its translocation to nucleus (Martina et al., 2012). Thus, it would be interesting to determine whether NINJ2 plays a role in modulating TFEB phosphorylation. In addition, the tumor suppressor p53 has been reported to engage in intricate crosstalk with TFEB to regulate basal autophagy (Suzuki et al., 2021). Interestingly, p53 expression is found to be increased by NINJ2-KO (Zhang et al., 2024a). It is, therefore, of interest to determine whether NINJ2 regulates TFEB activity through p53 or modulates the p53-TFEB signaling axis to maintain lysosomal integrity. Finally, NINJ1, the other member of the Ninjurin family, has been shown to promote pyroptosis. Our previous studies demonstrated that NINJ2 may antagonize NINJ1 by forming a complex with NINJ1. It will, therefore, be of considerable interest to determine whether NINJ1 also regulates LMP and whether it exhibits a role opposite to that of NINJ2.

Overall, our data expand the functional landscape of NINJ2 and establish it as a potential therapeutic node at the intersection of lysosomal integrity, iron metabolism, and ferroptosis.

Materials and methods

Reagents

Anti-FTH (Cat #4393 S; RRID:AB_11217441), anti-LAMP1 (Cat #3243 S, RRID:AB_2134478 and Cat #9091 S, RRID:AB_2687579), and anti-GAPDH (Cat #2118 L, RRID:AB_561053) were purchased from Cell Signaling Technology. Anti-actin (Cat #sc-8432, RRID:AB_626630) and anti-Galectin 3 (Cat #sc-32790, RRID:AB_627657) were purchased from Santa Cruz Technology. Anti-Flag (Cat #80801–2-RR, RRID:AB_3670488) and anti-FTL (Cat #10727–1-AP, RRID:AB_2278673) were purchased from Proteintech. Anti-NINJ2 was custom-made as previously described (Zhang et al., 2024c) and affinity purified. Goat anti-Rabbit IgG (FITC) (Cat #ab6717, RRID:AB_955238) and Goat Anti-Mouse IgG (FITC) (Cat #ab6785, RRID:AB_955241) were purchased from Abcam. Goat anti-Mouse IgG (Alexa 555) (Catalog # A-21422, RRID:AB_2535844) and Goat anti-Rabbit IgG (Alexa 555) (Cat # A-21429, RRID:AB_2535850) were purchased from Thermo Fisher Scientific. Proteinase inhibitor cocktail (Cat #78429), Trizol Reagent (Cat #15596026), RNAiMAX (Cat #13778), and RevertAid First Strand cDNA Synthesis Kit (Cat #K1621) were purchased from Life Technologies. The WesternBright Sirius HRP substrate (Cat #K12043-D20) was purchased from Advansta. JetPRIME transfection reagent (Cat #101000046) was purchased from Polyplus. Protein A/G magnetic beads (Cat # HY-K0202), RSL3 (Cat #HY-100218A), and Erastin (Cat # HY-15763) were purchased from MedChemExpress. QuantiChrom Iron Assay Kit (Cat # DIFE-250) was purchased from BioAssay Systems.

Cell culture

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MCF7, Molt 4, and 293T cells were purchased from the American Type Culture Collection (ATCC) and have been authenticated by ATCC. MCF7 and 293T were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Cat #12100061) supplemented with 10% fetal bovine serum (Gibco, Cat # 10437–028) and penicillin-streptomycin (Gibco, Cat #15140122). Molt4 cells were cultured in RPMI 1640 medium (Cat #11875093) supplemented with 10% fetal bovine serum and penicillin-streptomycin. Isogenic control and NINJ2-KO MCF7 and Molt4 cells were previously generated (Zhang et al., 2024b; Zhang et al., 2024a). Briefly, MCF7 and MOLT-4 cells were transfected with two gRNAs targeting NINJ2 and selected with puromycin for 3 weeks. Individual clones were then isolated, and NINJ2 knockout was confirmed by Western blot analysis. All the cells were used below passage 25 or within 2 months after thawing and tested free of mycoplasma.

Immunofluorescence microscopy

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MCF7 cells expressing 3 x Flag-tagged NINJ2 were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 in PBS, and blocked with 2% bovine serum albumin in PBS. The cells were then stained with primary antibodies, followed by fluorophore-conjugated secondary antibodies. The cells were mounted with ProLong Gold with DAPI and observed with a Leica SP8 confocal microscope with a 40 x oil immersion objective or 63x oil immersion objective. The dilutions of the primary antibodies were 1:100 for anti-LAMP1, 1:100 for anti-Ninj2, and 1:100 for anti-Gal3. The dilutions for the secondary antibody were 1:500 for Alexa 555-conjugated and 1:1000 for FITC-conjugated.

Lysosomal staining

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Cells were incubated with CytoFix Red Lysosomal stain (AAT, Cat# 23210) for 30 min at 37℃. The cells were fixed, permeabilized, and blocked, and then stained with primary antibodies followed by fluorophore-conjugated secondary antibodies as described above. The dilution for CytoFix Red was 1:500.

Proximity ligation assay (PLA assay)

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PLA assay was carried out by using the DuoLink PLA assay kit (Millipore Sigma, Cat# DUO92008, DUO92001, and DUO92005) according to the manufacturer’s protocol. Briefly, cells were fixed with 3.7% formaldehyde in PBS, permeabilized with 0.2% Triton X-100 in PBS, and blocked using the blocking reagent provided in the kit. The cells were then incubated with primary antibodies at 4 °C overnight (~20 hr). On the following day, cells were incubated with the PLUS and MINUS probes at 37 °C for 1 hr, followed by incubation with DNA ligase at 37 °C for 30 min and subsequent incubation with DNA polymerase together with the fluorescent probe at 37 °C for 100 min. The cells were mounted with ProLong Gold with DAPI and observed with a Leica SP8 confocal microscope as described above.

Transient transfection

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SiRNA transfection was performed with RNAiMAX according to the user’s manual. The sequence for scrambled siRNA was 5'-GCA GUG UCU CCA CGU ACU A-3’. The sequences for LAMP1 siRNA were 5'-CAG CAA UGU UUA UGG UGA AUU-3', and 5'-CCA AAG AAA UCA AGA CUG UUU-3'.

Western blot and immunoprecipitation (IP) analyses

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Western blot analysis was performed as previously described (Dohn et al., 2001). Briefly, whole-cell lysates were resolved on 8–13% SDS–polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated with primary and secondary antibodies, followed by detection using enhanced chemiluminescence and visualization with VisionWorks LS software (version 8.0; Analytik Jena, Jena, Germany). For IP analysis, Cells were lysed in an IP lysis buffer (1% NP-40, 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA) supplemented with protease inhibitor cocktail. The cell lysates with incubated with 1 μg of primary antibody and protein A/G magnetic beads at 4℃ overnight and the immunocomplexes were subjected to Western blot analyses to detect protein-protein association.

Cell viability

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4×10³ Cells were seeded per well in 96-well plates (100 μl per well) in quadruplicate and allowed to adhere overnight. Next day, cells were treated with varying concentrations of RSL3 or Erastin (0–7.29 μM) for 48 hr. Cell viability was assessed by adding 100 μl of CellTiter-Glo reagent (Promega) to each well and incubated for 10 min at room temperature. Following incubation, luminescence was measured using a SpectraMAX Gemini Microplate Reader (Molecular Devices, Silicon Valley, CA, USA). The viability in the control group was set as 100% and the relative cell viability was calculated as a percentage of treatment group vs control group. The IC₅₀ was calculated using GraphPad Prism 10.

Colony formation assay

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2×10³ Cells were seeded per well in a 6-well plate in triplicate. At 48 hr, cells were treated with various amounts of RSL3 or Erastin for 24 hr and the drug was withdrawn. Cells were then cultured in regular medium for 2 weeks to allow colonies to form. The colonies were then fixed with methanol/glacial acetic acid (7:1) and stained with 0.1% crystal violet at room temperature.

Total RNA isolation and RT-PCR analysis

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Total RNA was isolated with TRIzol reagent as described in the user’s manual (Life Technologies). 3 μg of total RNA was used to synthesize cDNA using the RevertAid First Strand cDNA Synthesis Kit, followed by PCR analysis. The program used for amplification was (i) 94 °C for 5 min, (ii) 94 °C for 45 s, (iii) 58 °C for 45 s, (iv) 72 °C for 30 s, and (v) 72 °C for 10 min. From steps 2–4, the cycle was repeated 28–35 times depending on the targets or 22 times for actin and GAPDH. The primers for LAMP1 were a forward primer, 5′- AGG ACA TAC ACT CAC TCT C-3′, and a reverse primer, 5′-GTG CCA CTA ACA CAT CTG-3′. The primers for FTH were a forward primer, 5′-CGA TGA TGT GGC TTT GAA GA-3’, and a reverse primer, 5′- AAT GGG GGT CAT TTT TGT CA-3′. The primers for HPRT1 were a forward primer, 5′-TAT GGC GAC CCG CAG CCC T-3′, and a reverse primer, 5′-CAT CTC GAG CAA GAC GTT CAG-3′. The primers for Actin were a forward primer, 5′-CTG AAG TAC CCC ATC GAG CAC GGC A-3′, and reverse primer, 5′-GGA TAG CAC AGC CTG GAT AGC AAC G-3′.

Labile iron measurements

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Cells were treated with ferric ammonium citrate (300 μg/ml) for 16 hr. After treatment, 1×10⁵ cells were collected and lysed in 200 μl of RIPA buffer. Cell lysates were then subjected to a labile iron assay using the QuantiChrom Iron Assay Kit according to the manufacturer’s instructions.

Protein half-life measurement

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Cells were mock-treated or treated with cycloheximide (50 μg/ml) from 0 to 15 hr. Cell lysates collected at each timepoint were subjected to Western blot analysis to detect FTH and actin. Band intensities at the different time points were quantified using the VisionWorks LS software, normalized to actin and plotted in a graph as a relative percentage of remaining protein.

Statistical analysis

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Student’s t-test was used for statistical analysis. P<0.05 is considered significant.

Data availability

All study data are included within the article.

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Article and author information

Author details

Jin Zhang

Department of Surgical and Radiological Sciences, University of California Davis School of Veterinary Medicine, Davis, United States

Contribution
Conceptualization, Data curation, Writing – original draft, Project administration, Writing – review and editing

For correspondence
jinzhang@ucdavis.edu

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-6835-920X
Miranda Bustamante

Department of Surgical and Radiological Sciences, University of California Davis School of Veterinary Medicine, Davis, United States

Contribution
Data curation

Competing interests
No competing interests declared
Yang Shi

Department of Surgical and Radiological Sciences, University of California Davis School of Veterinary Medicine, Davis, United States

Contribution
Data curation, Formal analysis

Competing interests
No competing interests declared
Ken-ichi Nakajima

Department of Surgical and Radiological Sciences, University of California Davis School of Veterinary Medicine, Davis, United States

Contribution
Data curation, Formal analysis

Competing interests
No competing interests declared
Xinbin Chen

Department of Surgical and Radiological Sciences, University of California Davis School of Veterinary Medicine, Davis, United States

Contribution
Conceptualization, Writing – original draft, Project administration, Writing – review and editing

For correspondence
xbchen@ucdavis.edu

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-4582-6506

Funding

National Institutes of Health (CA272753)

Xinbin Chen

National Institutes of Health (CA093373)

Xinbin Chen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported in part by National Institutes of Health R01 grants (CA272753), a UC Davis Cancer Center Core Support Grant (CA093373).

Version history

Sent for peer review: February 9, 2026
Preprint posted: February 11, 2026
Reviewed Preprint version 1: March 17, 2026
Reviewed Preprint version 2: August 19, 2026
Version of Record published: September 11, 2026

Cite all versions

You can cite all versions using the DOI https://doi.org/10.7554/eLife.110919. This DOI represents all versions, and will always resolve to the latest one.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.