CEMIP promotes extracellular matrix‐detached prostate cancer cell survival by inhibiting ferroptosis

Abstract Cells detached from the extracellular matrix (ECM) can trigger different modes of cell death, and the survival of ECM‐detached cells is one of the prerequisites for the metastatic cascade. Ferroptosis, a form of iron‐dependent programmed cell death, has recently been found to be involved in matrix‐detached cancer cells. However, the molecular mechanisms by which ECM‐detached cells escape ferroptosis are not fully understood. Here, we observed that cell migration‐inducing protein (CEMIP) upregulation facilitates ferroptosis resistance during ECM detachment by promoting cystine uptake in prostate cancer (PCa) cells. Meanwhile, silencing CEMIP causes it to lose its ability to promote cystine uptake and inhibit ferroptosis. Mechanistically, the interaction of CEMIP with inositol 1,4,5‐trisphosphate receptor type 3 (ITPR3) modulates calcium ion (Ca2+) leakage from the endoplasmic reticulum, activating calcium/calmodulin‐dependent protein kinase II (CaMKII), which further facilitates nuclear factor erythroid 2‐related factor 2 (NRF2) phosphorylation and nuclear localization, leading to elevated transcription of solute carrier family 7 member 11 (SLC7A11), a glutamate/cystine antiporter, in PCa cells. Our findings delineate a novel role of CEMIP in ferroptosis resistance during ECM detachment and provide new insights into therapeutic strategies for metastatic PCa.


| INTRODUC TI ON
Prostate cancer (PCa), with an estimated 1.4 million new cases and 375,000 deaths occurring in 2020, is the second most diagnosed malignant neoplasm in men worldwide. 1 Metastasis was found in 5% of PCa cases at first diagnosis, with a 5-year survival rate of <30%. 2,3 PCa cells in primary tumors often metastasize to the lymph nodes and distant organs, such as the bones, the lung, and the liver. These sites generally display terribly low responsiveness to initial treatment and have a high risk of post-treatment relapse.
Cancer cell survival after detachment from the extracellular matrix (ECM) is one of the prerequisites for the metastatic cascade.
Further, resistance to anoikis, a form of apoptosis induced by cell detachment, is crucial for the survival of ECM-detached cancer cells. 4 Growing evidence has shown that different modus of cellular changes in response to ECM detachment may also be involved in this procedure and induce cell death independent of anoikis. [5][6][7] Consequently, understanding the survival mechanisms in addition to anoikis resistance in ECM-detached cancer cells is essential for the development of effective chemotherapeutic strategies to inhibit tumor progression and metastasis.
Ferroptosis, a form of nonapoptotic cell death based on irondependent accumulation of reactive oxygen species (ROS), was first discovered by Dixon in 2012. 8 ECM detachment can cause a series of deleterious metabolic alterations, including a remarkable increase in ROS levels. 9 Therefore, ferroptosis may be activated when cells are detached from the ECM. Recent studies have shown that ECM detachment can trigger ferroptosis and that upregulation of the integrin α6β4 enables cancer cells to evade ferroptosis. 10 The cooperation of ΔNp63 with the B-cell lymphoma 2 protein inhibits oxidative stress-induced ferroptosis and confers clonogenic survival against matrix detachment. 11 However, whether matrix detachment triggers ferroptosis and how ECM-detached cells escape ferroptosis in PCa remains unclear.
Here, we demonstrate that ECM-detached PCa cells undergo ferroptosis, and detachment-resistant (DR) PCa cell models induced by continuous suspension culture exhibit tolerance to ferroptosis. Cell migration-inducing protein (CEMIP) is significantly overexpressed in DR PCa cell models and protects detached PCa cells against ferroptosis. Accumulating evidence suggests that CEMIP upregulation is related to cancer metastasis, cancer progression, and poor prognosis. [12][13][14][15] However, the effect of CEMIP on ferroptosis in metastatic PCa is unknown. In the present study, we aimed to discover the underlying regulatory mechanism of CEMIP in ferroptosis resistance during ECM detachment in PCa cells and to investigate the mechanisms involved.

| MATERIAL S AND ME THODS
The detailed procedures for the cell viability assay and measurement of intracellular ROS, malondialdehyde (MDA) and the ratio of reduced glutathione to oxidized glutathione (GSH/GSSG), patient tissue specimens, western blotting, nuclear and cytosolic protein extraction, quantitative real-time polymerase chain reaction (qRT-PCR), measurement of intracellular ferrous ion (Fe 2+ ) and calcium ion (Ca 2+ ), immunofluorescence staining, transmission electron microscope (TEM), plasmid transfection, soft agar assay, invasion and migration assays, luciferase reporter assay, co-immunoprecipitation (Co-IP), and animal studies are described in the Supplementary Materials and Methods.

| Cell culture and construction of the detachment-resistant cells model
PC-3, DU145, and LNCaP cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences. All cell lines were cultured in RPMI-1640 medium (Hyclone, GE Healthcare Life Sciences) containing 10% FBS (Biologic Industries) and 1% penicillin/streptomycin (Servicebio) under standard conditions (5% CO 2 , 37°C). The method used to construct the DR cells model was consistent that used in our previous studies. 16 In general, parental cells were suspension cultured in ultra-low-attachment six-well plates (Corning Life Sciences) for 7 days and then transferred to normal plates for continued adherent culturing for 2 days; the cells that re-adhered were considered DR cells.

Uptake of cystine was measured with BioTracker Cystine-FITC Live
Cell Dye (Sigma-Aldrich) using a previously reported method. 17 The pretreated cells were seeded on coverslips and incubated with F I G U R E 1 Death mechanism of PCa cells after detaching from extracellular matrix (ECM) involves ferroptosis. (A) CCK-8 assays depicting the change in cell viability of parental prostate cancer (PCa) cells were treated with DMSO or ZVAD-FMK (10 µM) or Ferrostatin-1 (5 µM) or ZVAD-FMK in combination with Ferrostatin-1 after suspended culture for 5 days. (B) Cell viability in parental and DR PCa cells were treated with DMSO or erastin (10 µM) or erastin in combination with Ferrostatin-1 (5 µM) for 24 h. (C) Intracellular ROS of parental and DR PCa cells were stained by DCFH-DA and determined by flow cytometry. (D, E) Concentrations of MDA and GSH/GSSH ratio were measured in parental and DR PCa cells. (F, G) Intracellular cystine and Fe 2+ of parental and DR PCa cells were detected by using cystine-FITC and FeRhoNox-1 fluorescent probe, respectively; confocal microscopy was used to record the fluorescence signal. Scale bars, 20 μm. (H) The morphological changes of mitochondria were detected by TEM in parental and detachment-resistant (DR) PCa cells. Scale bars represent 2.5 and 1 µm, respectively. Data are presented as representative images or as mean ± SD from three independent repeats. *p < 0.05, **p < 0.01, ***p < 0.001 complete medium for 24 h. After washing three times with choline chloride buffer, cells were starved at 37°C for 30 min in the same buffer. Then, cells were incubated with cystine-FITC (5 μM) in the dark for 60 min. Finally, cells were washed with choline chloride buffer three times and FITC fluorescence was detected using a Nikon A1Si Laser Scanning Confocal Microscope (Nikon Instruments).

| Immunohistochemistry
Immunohistochemical staining was undertaken as described previously, 16

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 9.0 software and were presented as the mean ± SD. All in vitro experiments were repeated three times. The differences between two groups were compared using Student's t-test. Comparisons among multiple sets of data were performed using ANOVA.

| Resistance to ferroptosis is required for survival of extracellular matrix-detached prostate cancer cells
To clarify whether ferroptosis is involved in the death mechanism of PCa cells after detachment from the ECM, we seeded parental PC-3 and DU145 cells in ultra-low-attachment 96-well plates. Cells from both cell lines were treated with the apoptosis inhibitor Z-VAD-FMK showed the most significant increase in cell viability (p < 0.001) ( Figure 1A). This indicates that the death mechanism of PCa cells after ECM detachment involves both apoptosis and ferroptosis.
To further investigate the influence of ferroptosis resistance during ECM detachment in PCa cells, we established DR cell models using PC-3 and DU145 cell lines, as described previously. 16 The CCK-8 assay showed that the ferroptosis inducer erastin (10 µM) reduced cell viability in both parental and DR cells. However, DR cells were significantly more resistant to erastin than parental cells. In addition, Ferrostatin-1 was able to reverse the effect of erastin on cell viability ( Figure 1B).
Next, we observed ferroptosis-related changes under nonadherent conditions in both parental and DR cells. ROS and MDA levels in DR cells were significantly lower than those in the parental cells ( Figure 1C,D). The ratio of GSH/GSSG and the uptake levels of cystine in DR cells were notably higher than those in their parental counterparts ( Figure 1E,F). No significant difference was observed in the levels of Fe 2+ accumulation among two groups ( Figure 1G). In addition, the differences in ferroptosis among parental and DR cells were also confirmed by transmission electron microscopy (TEM). As shown in Figure 1H, compared with DR cells, the mitochondria in parental cells were significantly smaller, while the mitochondrial cristae were shorter or had completely disappeared. These findings indicate that detachment from the ECM can lead to ferroptosis in PCa cells. Further, resistance to ferroptosis is required for the survival of PCa cells detached from the ECM.

| Cell migration-inducing protein mediates resistance to ferroptosis in DR prostate cancer cells
Using genome microarray assays, we previously found that CEMIP was significantly overexpressed in DR PCa cells compared to their parental cells. 16 Western blotting analysis and quantitative real- There were no significant differences in the intracellular Fe 2+ levels between groups ( Figure 2G). Consistent with these results, TEM showed that, compared to the NC group, CEMIP downregulation resulted in increased mitochondrial membrane density and smaller mitochondria ( Figure 2H). To fully understand the influence of CEMIP on ferroptosis in PCa cells, we also stably overexpressed CEMIP in parental PC-3 and DU145 cells. The results obtained were opposite to those obtained via CEMIP downregulation in DR cells, as shown in Figure S2. Collectively, downregulation of CEMIP attenuates resistance to ferroptosis in DR PCa cells. However, overexpression of CEMIP facilitates resistance to ferroptosis in parental PCa cells.

| Cell migration-inducing protein knockdown impairs antioxidant capacity of detachment-resistant prostate cancer cells by downregulating SLC7A11
As shown in Figure 3A,B, western blotting analysis and qRT-PCR suggested that, consistent with CEMIP, the expression of solute The expression of SLC7A11 was positively associated with the Gleason score of patients with PCa ( Figure 3G). Finally, the influence of SLC7A11 expression on the progress-free interval in PCa patients from TCGA was evaluated by univariate and multivariate Cox regression analysis. We found SLC7A11 to be significant in univariate Cox analysis but not significant in the multivariate Cox model, including SLC7A11, Gleason score, T stage, N stage, and M stage ( Figure 3H, Table S4). The baseline characteristics for PCa patients in the high and low SLC7A11 groups are shown in Table S5. Western blotting analysis also verified that SLC7A11 was upregulated in PCa tissues compared to matched normal tissues ( Figure 3I). Consistent with recent reports, 18 immunohistochemistry (IHC) analysis of PCa specimens also confirmed that SLC7A11 expression in PCa tissues was higher than that in corresponding pericarcinous specimens and that SLC7A11 H-scores were positively correlated with the Gleason scores in carcinoma tissues ( Figure 3J). These results suggested that SLC7A11 may be a major downstream effector of CEMIP. To test this hypothesis, we performed SLC7A11 rescue experiments in shCEMIP DR PCa and control cells. As shown in Figure S3   and is no longer degraded by ubiquitination. 24 Once phosphorylated, NRF2 translocates into the nucleus and activates the transcription of antioxidant response element (ARE)-containing genes. 25,26 As shown in Figure 5A Furthermore, the translocation of NRF2 between the cytoplasm and nucleus was confirmed by immunofluorescence staining ( Figure 5G).

| SLC7A11 rescues the progression of shCEMIP detachment-resistant prostate cancer cells in vitro and in vivo
To verify the transcriptional regulation of SLC7A11 by NRF2, we used JASPAR 27 (http://jaspar.gener eg.net) to identify the binding profile of NRF2 in Homo sapiens ( Figure 5H). Next, as shown in Figure 5I, we identified a canonical ARE upstream of the SLC7A11 transcription start site and cloned luciferase reporter constructs into a pGL3 plasmid containing the putative ARE (ARE-wt-luc) or its mutant (ARE-mut-luc). We also constructed NRF2(S40E) mutant plasmids, in which the S40 site was replaced with glutamic acid to mimic continual phosphorylation. 28 Subsequently, we performed a dual-luciferase assay to analyze promoter activity. As expected, ectopic NRF2(S40E) expression significantly activated ARE-wt-luc activity. However, no activation of ARE-mut-luc activity was observed upon overexpression of NRF2(S40E) ( Figure 5J). These results suggest that CEMIP promotes NRF2 phosphorylation and localization to the nucleus, which in turn promotes transcription of SLC7A11.

| Activation of calcium/calmodulin-dependent protein kinase II result in cell migration-inducing protein-mediated nuclear factor erythroid 2-related factor 2 phosphorylation at serine 40
The decreased phosphorylation level of NRF2 is accompanied by downregulation of CEMIP. However, CEMIP is a hyaluronidase rather than a classic protein kinase. 29 Studies have reported that detachment from the ECM activates endoplasmic reticulum (ER) stress, which leads to increased cytosolic Ca 2+ levels. 14,30 Recently, it was confirmed that CEMIP can interact with the ER receptor inositol 1,4,5-trisphosphate receptor type 3 (ITPR3), a key player in Ca 2+ signaling that mediates Ca 2+ leakage from the ER, leading to elevated cytosolic Ca 2+ levels. 31,32 To verify the interaction of CEMIP and ITPR3 in parental PCa cells and DR PCa cells, we performed a Co-IP experiment. As shown in Figure 6A,B, the interaction between CEMIP and ITPR3 in DR PCa cells was increased compared to that in parental cells. The cytosolic Ca 2+ levels in DR PCa cells were significantly higher than those in parental PCa cells ( Figure 6C). Furthermore, knockdown of CEMIP significantly decreased both the interaction between CEMIP and ITPR3 and the cytosolic Ca 2+ levels in DR PCa cells ( Figure 6D-F). Double immunofluorescent staining indicated that CEMIP and ITPR3 were colocalized in the cytoplasm of DR PC-3 cells. However, this colocalization relationship could be significantly weakened by knockdown of CEMIP ( Figure 6G).
To further address the effect of CEMIP on cytosolic Ca 2+ levels, we performed rescue experiments in parental PCa cells. As shown in Figure 6H, overexpression of CEMIP significantly rescued the inhibition of cytosolic Ca 2+ levels caused by the calcium chelator BAPTA-AM (1 µM). Based on the above research results, we further tested calcium/calmodulin-dependent kinase II (CaMKII) and its phosphorylation level at threonine 286 (T286), as CaMKII is a family of multifunctional serine/threonine protein kinases, and phosphorylated activation of CaMKII requires both Ca 2+ and calmodulin. 33,34 Western blotting analysis revealed that the phosphorylation level of CaMKII at T286 in DR PCa cells was remarkably higher than that in parental cells. However, there was no obvious difference in the expression of CaMKII between the two groups ( Figure 6I). Knockdown of CEMIP in DR PCa cells did not affect the expression of CaMKII but did reduce its activity ( Figure 6J). to cysteine and used to synthesize the major antioxidant GSH. 38,39 Given the difference in the ratio of GSH/GSSG and the uptake level of cystine between DR PCa cells and their shCEMIP group cells, we speculated that CEMIP might regulate the expression level of SLC7A11, which is the functional subunit of system X c − . 40 We found that CEMIP promotes ferroptosis resistance by facili- Cell migration-inducing protein localized in the ER is found to mediate ER Ca 2+ leakage and ultimately increase cytosolic Ca 2+ F I G U R E 5 Cell migration-inducing protein (CEMIP) drives NRF2 translocate from cytoplasm to nucleus and furthermore promotes the expression of SLC7A11. (A) KEAP1 protein, nuclear factor erythroid 2-related factor 2 (NRF2) protein, and its phosphorylation levels (p-NRF2) in the whole cell lysate from the indicated parental and detachment-resistant (DR) prostate cancer (PCa) cells were assessed using western blotting. (B) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the transcription levels of NRF2 in indicated parental and DR PCa cells. (C) Indicated parental PCa cells were subjected to nuclear and cytosolic fractionation, and NRF2 protein levels were determined by western blotting. Lamin B1 and β-Actin were used as nuclear and cytoplasmic markers, respectively. (D) The protein levels of NRF2, p-NRF2, and KEAP1 were assayed by western blotting in the indicated DR PCa cells transfected with empty plasmids or shCEMIP plasmids. (E) qRT-PCR to analyze changes in the transcription level of NRF2 caused by CEMIP knockdown in the indicated DR PCa cells. (F) The NRF2 protein levels in cytoplasmic fraction and nuclear fraction from the indicated cells were analyzed using western blotting. (G) NRF2 location in the indicated cells was observed using fluorescent microscopy. Scale bars, 20 μm. (H) The positional matrices for the human ARE sequences, JASPAR ID MA0150.1, which have been generated by the frequency at which these sites have been found experimentally to be occupied by NRF2. (I) Schematic representation of the ARE in the SLC7A11 promoter and its mutant (AREmut). The +1 denotes the transcription initiation site. (J) Luciferase assays performed using the control pGL3, ARE-wt, and ARE-mut constructs in the indicated parental PCa cells transfected with empty vector or NRF(S40E) plasmids. Data are presented as representative images or as mean ± SD from three independent repeats. ***p < 0.001 levels. 13  Data are presented as representative images or as mean ± SD from three independent repeats. **p < 0.01, ***p < 0.001 F I G U R E 7 A scheme for cell migrationinducing protein (CEMIP)-mediated ITPR3/CaMKII/NRF2/SLC7A11 pathway inhibits ferroptosis in detachmentresistant prostate cancer cells. CEMIP interacted with ITPR3 and promoted Ca 2+ leakage from the endoplasmic reticulum and thereby activated CaMKII. Activated CaMKII (p-CaMKII) further promoted the phosphorylation of NRF2, which led to NRF2 dissociated with KEAP1 and translocated from cytoplasm to nucleus. NRF2 facilitated SLC7A11 transcription and led to an increase in GSH levels, thereby inhibits ferroptosis