Collapsin response mediator protein 4 enhances radio-sensitivity through calcium-mediated cell signaling

Background: Radiation therapy, an effective treatment modality against various types of cancer including colorectal cancer, reduces local recurrence rate despite damaging both normal and cancer cells. However, the presence of cancer cells resistant to radiation therapy remains a major therapeutic obstacle; thus, understanding the mechanisms underlying radiation resistance is an important step toward achieving successful outcomes in cancer treatment. Hence, in the present study, radioresistant cell lines were established and the radiation-induced genetic changes associated with radiation resistance were examined. Methods: We generated radioresistant colorectal cancer cell lines and subjected them to RNA sequencing. To know the relationship between CRMP4 downregulation and radiation resistance, western blotting and ow cytometry were used. Results: CRMP4 was identied as the candidate gene associated with radiation sensitivity. The intracellular Ca 2+ concentrations increased when cells were exposed to radiation, which in turn, initiated apoptosis. Decreased CRMP4 expression enhanced resistance to radiation and Ca 2+ ionophore A23187. Conversely, Ca 2+ deciency by BAPTA-AM caused higher cell death in CRMP4-depleted cells than in CRMP4-expressing cells. Conclusion: Our results indicated that CRMP4 inuences Ca 2+ signaling pathways involved in apoptosis, and that CRMP4 is critical for radiation sensitivity in colorectal cancer as it can sensitize cancer cells to radiation therapy. genes in colorectal cancer. RNA-seq results showed CRMP4 downregulation in the radiation-resistant cell lines, compared to the CRMP4 expression level in their parental cell lines. Hence, the inuence of CRMP4 on radiosensitivity was investigated. Additionally, the role of CRMP4 during the response of irradiated CRC cells to Ca 2+ inux stress remains unclear. In the present study, the relationship between CRMP4 and Ca 2+ -mediated cell signaling was investigated to explain radiation sensitivity in CRC.

decreased apoptotic cell death rate and suppressed in ammatory responses [9]. CRMP4 is thus considered an important therapeutic target for neuroregeneration. In addition, several studies have indicated that CRMP4 is involved in various types of cancers. For example, pancreatic and colorectal cancers (CRCs) show elevated CRMP4 expression, which strongly correlates with severe venous invasion, liver metastasis, and poor prognosis [10,11]. Conversely, CRMP4 is regarded as a metastasis suppressor in prostate and breast cancer [12,13]. These results indicate that a deeper analysis of CRMP4 function may offer new insights into potential cancer therapies.
The mitochondrial membrane potential (MMP) is the major component of the proton-motive force, which is the central intermediate of aerobic energy production and the driving force behind other physiological processes in the mitochondria, such as Ca 2+ uptake and antioxidant activity [14]. Cellular injury or stress stimulation directly elicits alterations in mitochondrial architecture, membrane potential, and oxidative capacity, which are associated with an irreversible loss of mitochondrial matrix contents and integral membrane protein constituents such as cytochrome c oxidase [15]. The release of cytochrome c from the mitochondria leads to the activation of caspase-3 and -9, resulting in apoptosis [16]. In turn, Ca 2+ ions serve as an important second messenger for multiple physiological processes [17]. Several studies have indicated that intracellular Ca 2+ levels are regulated by ionizing radiation [18]; moreover, the rise in intracellular Ca 2+ levels after exposure to radiation is crucial for a diverse array of signaling pathways that regulate critical cellular processes including apoptosis [19,20]. Ca 2+ in ux is facilitated by voltageand ligand-gated Ca 2+ channels. Although CRMP4 has not been reported to be associated with Ca 2+ channels, CRMP2 was shown to interact with a novel N-type voltage-gated Ca 2+ channel [21,22]; nevertheless, the functional role of Ca 2+ binding to CRMPs remains elusive.
In the present study, the RNA-seq technique was used to investigate radioresistant-associated genes in colorectal cancer. RNA-seq results showed CRMP4 downregulation in the radiation-resistant cell lines, compared to the CRMP4 expression level in their parental cell lines. Hence, the in uence of CRMP4 on radiosensitivity was investigated. Additionally, the role of CRMP4 during the response of irradiated CRC cells to Ca 2+ in ux stress remains unclear. In the present study, the relationship between CRMP4 and Ca 2+ -mediated cell signaling was investigated to explain radiation sensitivity in CRC.

Radiation and radioresistant cell line generation
Cells were seeded in 60-mm dishes and exposed to radiation from a 60 Co source (model 109 irradiator; JL Shepherd and Associates, San Fernando, CA, USA) at the indicated doses (0-5 Gy). Subsequently, cells were incubated at 37 °C under a humidi ed, 5% CO 2 / air atmosphere.
To generate radiation resistance CRC [23,24] using SW620, RKO, SW480, and HT-29 cell lines, CRC cells were plated in 60-mm dishes at a density of 5 × 10 3 cells/dish and exposed to a 5-Gy dose of ionizing radiation, followed by a 15-day recovery period. This process was repeated for 24 treatment cycles totaling 120 Gy; nally, radioresistant IR-SW620, IR-RKO, IR-SW480, and IR-HT-29 cells lines were established.
Flow cytometry for the measurement of MMP, intracellular calcium levels, and apoptosis assay The uorescent probe Fluo 3-AM was used for the assessment of intracellular levels of Ca 2+ and the lipophilic cationic dye 3,3¢-dihexyloxacarbocyanine iodide (DiOC6(3)) was used for measuring disruption of the MMP (Δψm). Brie y, cells were exposed to a 5-Gy radiation dose and incubated for 72 h, then stained with 1 μM DiOC6(3) at 37 °C for 15 min in the dark, and analyzed using FACSverse ow cytometry (BD Biosciences, San Jose, CA, USA). For apoptosis analysis, cells were harvested and centrifuged at 800 rpm for 3 min following radiation treatment (48 h). Cells were carefully resuspended, then annexin-V (5 μL) and propidium iodide (PI) (5 μL) (BD Biosciences) were added and the cells incubated at 37 °C for 15 min in the dark. The stained cells were analyzed using FACSverse ow cytometry.

Clonogenic assay
Cells were seeded into 60-mm dish plates at densities of 1, 2, 5, 8, and 10 × 10 3 cells/plate. After 24 h, cells were radiated with the indicated dose of ionizing radiation. After 14 days, the colonies were subsequently xed and stained with 0.1% crystal violet in 20% ethanol and were counted. Alternatively, the remaining attached cells were stained with crystal violet; after a wash step, the dye was solubilized and absorbance measurements at 590 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA) were taken.

Cell-cycle analysis
Irradiated cells were trypsinized, washed in ice-cold PBS, and xed with 70% ethanol on ice. Fixed cells were stained with PI (BD Biosciences) containing RNase (0.1 mg/mL) for 15 min at 37 °C, and cell population analysis was performed using a FACSCalibur ow cytometry.

Measurement of cytochrome c release
The cytochrome c-releasing apoptosis assay kit (Abcam ab65311, Cambridge MA, USA) was used for detecting cytochrome c translocation from mitochondria into the cytosol. Irradiated or A23187-treated cells were lysed in a cytosolic extraction buffer, homogenized, the supernatant cytosol fraction separated by centrifugation, and the pellet resuspended in a mitochondrial extraction buffer, according to the manufacturer's instructions. Separated cytosolic and mitochondrial fractions were immunoblotted for the measurement of cytochrome c release.
Cell-viability assay Cell viability was assessed using the water-soluble tetrazolium salt (WST)-1 assay (Roche, Mannheim, Germany) according to the manufacturer's instructions. Brie y, 10 μL WST-1 reagent was added to each well of a 96-well plate (1 × 10 4 cells/well). After incubation for 1 h, the conversion of WST-1 reagent into chromogenic formazan was evaluated using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Statistical analysis
All results were con rmed in at least three independent experiments; and data from one representative experiment is shown. All quantitative data are presented as the means ± standard deviation (SD); Student's t-tests and ANOVA were used for comparisons of means of quantitative data between groups; a value of p < 0.05 was considered statistically signi cant.

CRMP4 expression was downregulated in radioresistant CRC cell lines
To identify genes associated with radiation-induced cell death, radioresistant IR-SW620, IR-RKO, IR-SW480, and IR-HT-29 cells lines were established as described above. CRC cells were exposed to ionizing radiation over several weeks for a total exposure of 120 Gy [23,24]. Thereafter, the surviving cells were selected and characterized using radiation resistance that was lacking in parental cells. From the RNA sequencing analysis, a total of 25,207 genes were identi ed. Among them, 70 genes were upregulated (>1.5 fold), while 45 genes were downregulated (<0.5 fold). These 115 genes were con rmed using RT-PCR analysis. Finally, the upregulated genes (CXCR4, RAC2, HBE1 [23], PTGDS, and LCN2) and downregulated genes (SMO, RGS10, PRTFDC1, and CRMP4) were identi ed as candidate genes associated with radiation resistance. It was determined that collapsin response mediator protein 4 (CRMP4) was signi cantly downregulated. Western blot analysis showed reduced CRMP4 expression in radiation-resistant cells (IR-SW480, IR-SW620, IR-RKO) compared with the expression level observed in parental cells (Fig. 1a). This result implied that CRMP4 may act as a radiation sensitivity-associated gene.
Since CRMP4 expression is signi cantly elevated in the SW620 and RKO cell lines, these cells were chosen as our experimental model. IR-resistant cell lines were established with acquired resistance to radiation. A clonogenic assay was performed to con rm the establishment of IR-SW620 and IR-RKO cell lines (Fig. 1b). The degree of apoptosis was monitored following radiation. Cells irradiated with a 5-Gy radiation dose and then stained with annexin V and PI were analyzed using a FACSverse ow cytometer. Apoptosis was reduced in the IR-SW620 and IR-RKO cells when compared with the degree of apoptosis seen in parental cells (Fig. 1c). To investigate the effect of radiation on the cell cycle, PI staining was employed. It was found that the cells were arrested at the G2/M phase following radiation, probably due to DNA damage. The radiation-induced G2/M arrest was reduced in both IR-SW620 and IR-RKO cells compared to the corresponding rate of cell-cycle arrest in parental cells (Fig.1d). These cell lines were thus determined to be well suited to radiation resistance mechanistic studies.

CRMP4 knockdown by siRNA augmented radiation resistance
Loss-of-function experiments using siRNAs were performed on CRC cell lines to determine whether CRMP4 reduction was involved in radiation resistance. First, CRC cell lines SW480, Caco-2, and KM12C, all of which expressed CRMP4, were selected and transfected with CRMP4 siRNA. The clonogenic assays revealed that CRMP4 depletion was associated with resistance to radiation in these cells (Fig. 2a). Next, it was examined whether increased clonogenic survival by CRMP4 knockdown was associated with reduced apoptosis. Annexin V staining was used to investigate the degree of apoptosis induced by radiation in CRMP4 knockdown cells and mock cells. The number of apoptotic cells following radiation in CRMP4 knockdown cells was less than that found in mock cells transfected with negative-control siRNA (Fig. 2b). This implied that CRMP4 depletion was associated with greater resistance to radiation. In the FACS cell-cycle analysis, CRMP4 depletion decreased the extent of G2/M arrest associated with radiation (Fig. 2c). Collectively, these ndings indicated that the suppression of CRMP4 signi cantly contributes to the development of radiation resistance in CRC cells.
CRMP4 depletion attenuated radiation-induced cytochrome c release from mitochondria Irradiation has been shown to induce various cellular and molecular damage outcomes, including apoptosis, in which cytochrome c release from mitochondria constitutes a critical event [25]. The amount of cytochrome c released from the mitochondria of irradiated cells was measured using a mitochondria/cytosol fractionation kit. Irradiated cells induced cytochrome c release in a time-dependent manner. The maximum amount of cytochrome c release was attained at 72 h of exposure to 5 Gy of radiation. Fig. 3a shows that decreasing amounts of cytochrome c were detected in the cytosol of the radiation-resistant IR-SW620 cells compared to the level in the cytosol of parent cells. These differences affect cell susceptibility to radiation.
To investigate CRMP4 action in CRC cells, CRMP4 expression was stably knocked down in SW620 and RKO cells using lentiviral vectors. Next, the effect of CRMP4 on cytochrome c release was evaluated following radiation exposure. Fig. 3b shows that the loss of CRMP4 expression was associated with reduced cytochrome c release and PARP cleavage. Previous studies have indicated that the release of cytochrome c from mitochondria during apoptosis is associated with low MMP (ΔΨm). The lipophilic cationic dye 3DiOC6(3) was used to monitor MMP and determine whether CRMP4 reduction was associated with MMP loss. Cells were irradiated, and MMP was measured over time. IR-resistant cells showed decreased mitochondrial depolarization compared to the level of depolarization seen in parental cells (Fig. 3c). Likewise, CRMP4 absence was associated with a reduction in the radiation-induced MMP collapse when compared with mock cells (Fig. 3d). These results indicated that CRMP4 absence can mitigate MMP collapse following exposure to radiation.
CRMP4 depletion attenuated calcium-mediated cell death signaling Consequent to its role as an incredibly versatile signaling ion, uncontrolled cytosolic Ca 2+ in ux induces mitochondrial dysfunction and cell death pathways [4,20]. Intracellular Ca 2+ concentrations were measured using the cell-permeable uorescent Ca 2+ indicator, Fluo 3-AM, in ow cytometry to determine whether CRPM4 expression could modulate Ca 2+ in ux. Increased intracellular Ca 2+ concentrations were observed in all irradiated cells irrespective of CRMP4 expression. (Fig. 4a). These data indicated that CRMP4 is not involved in intracellular Ca 2+ in ux regulation. Nonetheless, the in ux of intracellular Ca 2+ by radiation is associated with apoptosis. Excessive intracellular Ca 2+ concentrations can also cause the mitochondrial permeability transition (MPT) pore to open. The MPT pore is interrupted by cyclosporin A (CsA). When clonogenic assays were performed, CsA prevented radiation-induced apoptosis (Fig. 4b).
Based on these results, it was proposed herein that Ca 2+ is critical to radiation-induced cell death.
It was further analyzed whether CRMP4 is required for Ca 2+ -mediated apoptosis signaling using the Ca 2+ ionophore A23187, which boosts intracellular Ca 2+ levels. The WST-1 assay results revealed that CRMP4depleted cells and IR-resistant cells signi cantly reduced A23187-induced cell death in a dose-dependent manner compared to parent cells (Fig. 4c). Thereafter, the mitochondrial release of cytochrome c into the cytosol was analyzed following A23187 treatment; it was determined that the levels of cytosolic cytochrome c following treatment with A23187 decreased in CRMP4-depleted cells compared with the corresponding levels in mock cells (Fig. 4d). Likewise, under the same experimental conditions, decreased mitochondrial depolarization was observed in CRMP4-depleted cells. (Fig. 4e). It was theorized that these differences in the degree of cytochrome c release and mitochondrial depolarization may be associated with CRMP4 expression in Ca 2+ -mediated cell death signaling.

Ca 2+ inhibitor reduced the CRMP4 level and enhanced radioresistance
To evaluate the role of Ca 2+ signaling in radiation-induced apoptosis, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell-permeant intracellular Ca 2+ chelator that is used in manipulating intracellular Ca 2+ levels, was employed. In previous experiments, the inhibition of MPT by CsA reduced CRMP4 expression. Therefore, it was con rmed that CRMP4 expression was altered by intracellular Ca 2+ levels. Interestingly, CRMP4 expression in CRC cell lines treated with BAPTA-AM was also decreased in a dose-dependent manner (Fig. 5a). However, the reduction in CRMP4 expression by BAPTA-AM warrants further investigation. BAPTA-AM is commonly used to form Ca 2+ buffers with well-de ned Ca 2+ concentrations. Thus, BAPTA-AM maintains intracellular Ca 2+ homeostasis. The buffering of intracellular Ca 2+ concentrations by BAPTA-AM also reduced A23187-induced release of cytochrome c in SW620 cells (Fig. 5b). Western blot results showed that cleaved-PARP was augmented following radiation exposure; conversely, its level was diminished following treatment with radiation and the Ca 2+ chelator BAPTA-AM (Fig. 5c). Likewise, BAPTA-AM prevented radiation-induced mitochondrial depolarization in both mock and CRMP4-depleted cells (Fig. 5d). These data indicated that treatment with the cytosolic Ca 2+ chelator BAPTA-AM can alleviate A23187-and radiation-induced cell damage. This data suggest that radiation-induced cell death is mediated by cytosolic Ca 2+ signaling and Ca 2+ homeostasis, which are both essential for cell survival.

Ca 2+ de ciency-induced cell death was inhibited via CRMP4
The functional signi cance of CRMP4 in Ca 2+ signaling was investigated. Several studies have demonstrated the extensive use of BAPTA-AM for buffering Ca 2+ , however, high concentrations of BAPTA-AM have been found to deplete intracellular Ca 2+ stores [11,26]. Cells were treated with a high concentration of BAPTA-AM to assess the Ca 2+ de ciency-induced cell death rate; the surviving cells were stained with crystal violet dye (Fig. 6a). Also, WST-1 assays were performed for cell-viability measurement, as shown in Fig. 6b. These results showed that BAPTA-AM-induced Ca 2+ de ciency leads to cell death in both IR-resistant and CRMP4-depleted cells in a dose-dependent manner, but no signi cant effect was observed in parental cells. This indicated that Ca 2+ de ciency-induced apoptosis depends on the CRMP4 expression levels. As anticipated, cleaved-PARP was elevated in both IR-resistant and CRMP4-depleted cells treated with BAPTA-AM in a dose-dependent manner, but not in parental cells (Fig. 6c). Based on these results, it was hypothesized that CRMP4 is important for survival during cellular stress responses to BAPTA-AM-induced Ca 2+ de ciency, and that it is involved in Ca 2+ signaling.

Discussion
Previously, γ-irradiation-resistant CRC cell lines were established and novel candidate molecules implicated in radioresistance were identi ed using RNA sequencing analysis [23]. Among the genes signi cantly downregulated in radioresistant cell lines than in their parent cells, CRMP4 was further examined in the present study with regard to the association between tumor growth and radioresistance. CRMPs in uence various intracellular signal transduction pathways including VEGF, RhoA, GSK3β, and sema3A [27,28]; moreover, CRMP4 is involved in neurodevelopmental disorders such as schizophrenia, neurological disorders such as Alzheimer's disease [29], and various types of cancers such as breast, prostate, gastric, and hepatocellular carcinomas [12,[30][31][32][33].
Ca 2+ constitutes a ubiquitous diffusible intracellular second messenger that is released inside cells upon ligand interaction with membrane receptors; it is especially associated with diverse cellular functions related to cell growth but can induce apoptosis. A primary cause of Ca 2+ -induced mitochondrial damage is the nonspeci c pore opening of the mitochondrial membrane leading to the activation of MPT, which causes loss of MMP, rupture of the outer mitochondrial membrane, and leakage of intermembrane proteins, such as cytochrome c, to the cytoplasm [34][35][36]. Therefore, the disruption of Ca 2+ homeostasis in cells affects various signaling pathways including those associated with proliferation and apoptosis [16,26]. It was recently reported that patients with Hodgkin's lymphoma who receive a total dose of more than 30 Gy of radiotherapy displayed signi cantly higher Ca 2+ scores than other patients, putting them at a higher risk of coronary artery disease [37]. Nonetheless, our understanding of the correlation between radiation and Ca 2+ homeostasis remains insu cient.
Our results showed that radiation-induced intracellular Ca 2+ in ux increased similarly in both mock and CRMP4-depleted cell lines. However, CRMP4 enhances sensitivity to radiation. Likewise, A23187-induced cell death is decreased in CRMP4-depleted cells compared with the cell death rate in mock cells. Based on these results, it can be inferred that CRMP4 plays a role in Ca 2+ signaling pathways involving apoptosis in CRC cells. In addition, the cellular effects of BAPTA-AM, a cell-permeant intracellular Ca 2+ chelator that acts as an intracellular Ca 2+ buffer, on CRC cells were investigated. Western blot results showed that BAPTA-AM was associated with increased radiation resistance in cancer cells. Cleaved-PARP levels in radiosensitive parental cells were signi cantly reduced upon BAPTA-AM treatment (5 μM). These results implied that Ca 2+ signi cantly in uences radiation-induced apoptosis. It was also revealed that the depletion of intracellular Ca 2+ induced by high concentrations of BAPTA-AM (10-20 μM) was associated with a lower cell survival rate in CRMP4-depleted cells, but not in CRMP4-expressing cells. According to previous studies, several intracellular protein transport systems can be affected by the chelation of Ca 2+ with BAPTA-AM [38], potentially including vesicle formation. It was therefore predicted herein that CRMP4 could be involved in Ca 2+ signaling, since CRMP4 activity would be essential for cell survival during intracellular Ca 2+ de ciency.
Our results also indicated that CRMP4 expression levels were altered by several factors, including CsA and BAPTA-AM, implying that CRMP4 may be an adjustable target protein. It has been reported that CRMP4 expression levels are regulated by miRNAs. miR-130a upregulation has been reported to target the 3'UTR region of CRMP4 in gastric cancer cells and promote tumor progression via CRMP4 inhibition [39]. Conversely, VEGF enhances CRMP4 expression levels in gastric cancer cells, which is inhibited by the MAPK inhibitor, PD98059, and the PI3K inhibitor, LY294002. In mice, CRMP4 overexpression facilitates tumor growth and metastasis [40]. Taken together, these results implied that CRMP4 can be controlled and that the modulation of CRMP4 expression at the cellular level may affect cancer-cell radiation sensitivity.

Conclusion
In conclusion, it was con rmed herein that the CRMP4-mediated Ca 2+ signaling pathway is critical for radiation sensitivity in colorectal cancer. CRMP4 thus serves as a potential target for the sensitization of colorectal cancer cells to radiotherapy. Additionally, it was con rmed that intracellular Ca 2+ levels are an in uential factor in colorectal cancer radiation therapy.

Declarations
Ethics approval and consent to participate Not applicable.
Consent for publication     CRMP4 plays a role in the Ca2+ -mediated cell death signaling pathway. (a) Mock cells and CRMP4depleted cells were treated with 0 or 5 Gy of radiation. After 74 h, cells were loaded with Fluo 3-AM and the intracellular Ca2+ level was measured by ow cytometry. N.S, not signi cant, compared with control cells. (b) SW620 cells were exposed to a 5-Gy radiation dose in the presence/absence of CsA (cyclosporin A) (5 μM, 1 h before). After 14 days, cells were stained with 0.1% crystal violet. Representative images showed surviving cells (c) Cells were treated with an increasing dose of A23187. After 24 h, cells were incubated with WST-1 (water-soluble tetrazolium salt-1) reagent and the absorbance was measured with a microplate reader. Cell viability is expressed as the percentage of control cells. *P < 0.05, **P < 0.01. (d) Cells were incubated with the indicated doses of A23187 for 12 h. The cytosolic fractions were isolated and the contents of cytochrome c and CRMP4 were examined by western blot analysis. (e) Mock cells and CRMP4-depleted cells were treated with the indicated doses of A23187. After 24 h, mitochondrial membrane potential was analyzed by ow cytometry Figure 5 Blocking the rise of intracellular Ca2+ enhances radioresistance. (a) SW620 cells were treated with either indicated doses of BAPTA-AM or A23187 for 24 h and CRMP4 expression was analyzed by western blot analysis. (b) SW620 cells were treated with A23187 (+, 1 μM; ++, 2 μM) in the presence/absence of 5 μM BAPTA-AM for 12 h. Mitochondrial and cytosolic fractions were extracted for western blot analysis. (c) SW620 and IR-SW620 cells were treated with (or without) 5 μM BAPTA-AM before a 5-Gy radiation dose. After 72 h, the levels of C-PARP (cleaved-poly ADP-ribose polymerase) and CRMP4 were detected by western blot analysis. (d) Cells were exposed to 5 Gy of radiation in the presence/absence of 5 μM BAPTA-AM. After 72 h, mitochondrial membrane potential was analyzed by ow cytometry