Disrupting CISD2 function in cancer cells primarily impacts mitochondrial labile iron levels and triggers TXNIP expression

Abstract

The CISD2 (NAF-1) protein plays a key role in regulating cellular homeostasis, aging, cancer and neurodegenerative diseases. It was found to control different calcium, reactive oxygen species (ROS), and iron signaling mechanisms. However, since most studies of CISD2 to date were conducted with cells that constitutively lack, overexpress, or contain mutations in CISD2, the relationships between these different signaling processes are unclear. To address the hierarchy of signaling events occurring in cells upon CISD2 disruption, we developed an inducible system to express CISD2, or the dominant-negative H114C inhibitor of CISD2, in human breast cancer cells. Here, we report that inducible disruption of CISD2 function causes an immediate disruption in mitochondrial labile iron (mLI), and that this disruption results in enhanced mitochondrial ROS (mROS) levels. We further show that alterations in cytosolic and ER calcium levels occur only after the changes in mLI and mROS levels happen and are unrelated to them. Interestingly, disrupting CISD2 function resulted in the enhanced expression of the tumor suppressor thioredoxin-interacting protein (TXNIP) that was dependent on the accumulation of mLI and associated with ferroptosis activation. CISD2 could therefore regulate the expression of TXNIP in cancer cells, and this regulation is dependent on alterations in mLI levels.

Keywords: CISD2; Cancer; Ferroptosis; Iron homeostasis; Iron-sulfur cluster [Fe–S]; Mitochondria; NAF-1; Oxidative stress; Reactive oxygen species (ROS); TXNIP.

Figure 1.

Inducible expression of H114C suppresses the growth rate of human epithelial breast cancer cells. A-C. Protein blot analysis of CISD2 expression in Control (A), CISD2 (B) and H114C (C) cells upon induction of the Tet-One inducible expression system using Doxycycline (DOX) (2μg/ml). Representative protein blots are shown on top and β-actin-normalized expression graph are shown on bottom. Results in (A-C) are shown as mean ± SE of 5 biological control averages, each conducted with 3 technical repeats. ***P<0.001; N=5; one sample t-test. D-E. Cellular viability (D) and growth rate (E) of Control-, CISD2- and H114C-expressing cells 72 hours following the induction of protein expression with DOX (0, 0.5, 1.0, 1.5, 2.0, 2.5 μg/ml). Cell viability was measured using Alamar blue (D), and cell growth was measured with an Incucyte® Live-Cell Imaging apparatus (E). Results in (D-E) are shown as mean ± SE of 4 biological control averages, each conducted with 3 technical repeats. N=4; two-way ANOVA followed by a Tukey test; ns, not significant.

Figure 2.

Accumulation of mitochondrial labile iron (mLI) and mitochondrial ROS (mROS) following Doxycycline (DOX)-induced expression of H114C in cancer cells. A. Quantitative analysis of mLI accumulation in H114C cells upon DOX (2μg/ml)-induced protein expression in Control, CISD2 and H114C cells. Quenching of RPA fluorescence indicates mLI accumulation. B. Quantitative analysis of mROS accumulation in Control, CISD2 and H114C cells, upon DOX (2μg/ml)-induced protein expression. Results in A and B are mean ± SE of N=10 (A), or N=6 (B), biological control averages, each conducted with 3 technical repeats; two-way ANOVA followed by a Tukey test; ns, not significant.

Figure 3.

Alterations in cytosolic and ER calcium levels following Doxycycline (DOX) (2μg/ml) -induced expression of H114C in cancer cells. A. Decreased cytosolic calcium levels in H114C cells upon DOX (2μg/ml)-induced protein expression in control, CISD2 and H114C cells. B. Enhanced accumulation of ER calcium levels in H114C cells upon DOX (2μg/ml)-induced protein expression in Control, CISD2 and H114C cells. C. Levels of mitochondrial calcium levels upon DOX (2μg/ml)-induced protein expression in Control, CISD2 and H114C cells. Results are shown as mean ± SE of 5 biological control averages, each conducted with 3 technical repeats. N=5; two-way ANOVA followed by a Tukey test; ns, not significant.

Figure 4.

Application of the mitochondria-permeable iron chelator Deferiprone (DFP) suppresses mitochondrial labile iron (mLI) and mirochondrial ROS (mROS) accumulation but has no effect on changes in ER and cytosolic calcium levels. A. Application of DFP suppresses the accumulation of mLI levels in DOX (2μg/ml; 48 hour)-treated H114C cells. B. Application of DFP suppresses the accumulation of mROS levels in DOX (2μg/ml; 48 hour)-treated H114C cells. C. Application of DFP does not affect the decrease in cytosolic calcium levels in DOX (2μg/ml; 72 hour)-treated H114C cells. D. Application of DFP does not affect the increase in ER calcium levels in DOX (2μg/ml; 72 hour)-treated H114C cells. Results are shown as mean ± SE of 6, 7 or 12 biological control averages, each conducted with 3 technical repeats. N=6 for (A), N=7 for (B), and N=12 for (C and D); two-way ANOVA followed by a Tukey test; ns, not significant.

Figure 5.

Time-course proteomics analysis of Control-, CISD2- and H114C-expressing cells following application of Doxycycline (DOX; 2μg/ml) for 0, 3, 6, 12, and 24 hours. A. Venn diagram showing the overlap between proteins significantly altered in their expression in Control, CISD2 and H114C cells upon treatment with DOX (2μg/ml) for 0, 3, 6, 12 and 24 hours. B. Heatmap showing the expression pattern of the 118 proteins uniquely expressed following the application of DOX (2μg/ml) for 0, 3, 6, 12, and 24 hours to H114C cells. C. Venn diagram and line graphs showing the expression pattern of iron-, oxidative stress- and calcium- response proteins in H114C cells following the application of DOX (2μg/ml). Please see Supplementary Tables S1–S6, Supplementary Figure 3, and text for additional details. All proteins shown were found to be significantly expressed (P<0.05; N=3) compared to time 0 hour by a one-way Welch ANOVA.

Figure 6.

Expression of thioredoxin-interacting protein (TXNIP) and mitoNEET (mNT) following the application of Doxycycline (DOX; 2μg/ml). Expression of the tumor suppressor TXNIP (A) or the anti-ferroptosis ([2Fe-2S] CISD1) protein mNT (B) following the application of Doxycycline (DOX; 2μg/ml) to Control, CISD2 and H114C cells for 0, 3, 6, 12, and 24 hours. Results are shown as mean ± SE of 7 (A) or 5 (B) biological control averages, each conducted with 3 technical repeats. ***P<0.001; N=7 (A); N=5 (B); one sample t-test.

Figure 7.

Detection of ferroptosis markers in cancer cells following Doxycycline (DOX; 2μg/ml)-induced expression of the H114C protein and suppressed expression of thioredoxin-interacting protein (TXNIP) in H114C cells upon application of the mitochondria-permeable iron chelator Deferiprone (DFP). A-C. Expression of glutathione peroxidase 4 (GPX4; A), transferrin receptor (TfR; B), and mitochondrial thioredoxin 2 (TRX; C) in H114C cells following induction of H114C expression with DOX (2μg/ml) for 0, 24, 48, and 72 hours. Results in (A-C) are shown as mean ± SE of 7 biological control averages, each conducted with 3 technical repeats. **P<0.01, ***P<0.001; N=7; one sample t-test. D. Lipid peroxidation in Control, CISD2 and H114C cells 72 hours following DOX (2μg/ml) application. Results in are shown as mean ± SE of 6 biological control averages, each conducted with 3 technical repeats. *P<0.05, ***P<0.001; N=7; Student’s paired t-test. E. Expression of TXNIP in Control, CISD2 and H114C cells 24 hours following induction with DOX (2μg/ml) in the presence or absence of DFP. Results in are shown as mean ± SE of 6 biological control averages, each conducted with 3 technical repeats. ***P<0.001; N=7; one sample t-test.

Figure 8.

Treatment with Ferrostatin-1 mitigates the negative effects of H114 expression on cell viability and growth. A. The effect of Ferrostatin-1 treatment (2μM) on cell viability of Doxycycline (DOX; 0, 0.5, 1.0, 1.5, 2.0, 2.5 μg/ml)-treated Control (Upper), CISD2 (Middle) and H114C (Lower) cells at 72 hours following DOX application (with or without Ferrostatin). Cell viability was measured using Alamar Blue. B. The effect of Ferrostatin-1 treatment (2μM) on cell growth of Doxycycline (DOX; 0, 0.5, 1.0, 1.5, 2.0, 2.5 μg/ml)-treated Control (Upper), CISD2 (Middle) and H114C (Lower) cells at 72 hours following DOX application (with or without Ferrostatin). Cell growth was measured with an Incucyte® Live-Cell Imaging apparatus. Results in A and B are shown as mean ± SE of 7 biological control averages, each conducted with 3 technical repeats. N=7; two-way ANOVA followed by a Tukey test; ns, not significant.