Schlafen Family Intra-Regulation by IFN-α2 in Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) has a poor prognosis and no targeted therapy for treatment. The Schlafen gene family, particularly SLFN12, critically mediates TNBC biology. Higher expression of SLFN12 correlates with decreased TNBC viability and increased chemosensitivity and patient survival, yet no treatment is known to upregulate SLFN12 in TNBC. We hypothesized that Interferon-α (IFN-α2) upregulates SLFN12 in TNBC, subsequently reducing cell viability. We utilized short hairpin adenovirus to knockout SLFN12 (AdvShSLFN12) in MDA-MB-231, Hs-578T, and BT-549 TNBC cells. Cells were treated with AdvShSLFN12 and IFN-α2. After treatment, TNBC cell viability, SLFN family mRNA, and protein expression were analyzed. Treating TNBC cells with IFN-α2 increased SLFN12 expression and reduced cell viability. However, when AdvShSLFN12 knocked down SLFN12 during IFN-α2 treatment, TNBC cell viability was still reduced. We, therefore, investigated the potential involvement of other SLFN members IFN-α2 effects on cell viability. IFN-α2 increased SLFN5, SLFN12-Like, and SLFN14 but not SLFN11 or SLFN13. During AdvShSLFN12 + IFN-α2 treatment, the expressions of SLFN5, SLFN12-Like, and SLFN14 further increased. However, when siRNA knocked down SLFN5, SLFN12-Like, and SLFN14, the IFN-α2 reduction in viability was blunted. Although the interpretation of these results may be limited by the potential interactions between different siRNAs, these data suggest a complex regulatory signaling cascade among SLFN family members. Targeting this cascade to manipulate SLFN levels may, in the future, offer the potential to manipulate the chemosensitivity of TNBC tumors.

Keywords: AdvShSLFN12; IFN-?2; Schlafen 12; TNBC; cell viability.

Figure 1

IFN-𝛼2 increases SLFN12 expression, whereas AdvShSLFN12 prevents the IFN-𝛼2-associated increase in SLFN12. (A) mRNA analysis by primer-probe RT-qPCR confirmed that the adenovirus short hairpin RNA for SLFN12 (AdvshSLFN12) is effective at knocking down SLFN12 in MDA-MB-231 cells after 48 h (n = 6, p < 0.0001). -qPCR analysis following treatment with IFN-𝛼2 was able to induce SLFN12 but not in the presence of AdvshSLFN12 after 48 h in (B) MDA-MB-231 (n = 6, p < 0.0001), (C) BT-549 (n = 3, p < 0.001), and (D) Hs-578T (n = 3, p < 0.0001). RPLP0 was used as a reference gene. All error bars shown represent the standard error of the mean. Asterisks denote significance between control and each condition, whereas crosses indicate significance between shown conditions. p-value is for both asterisks and crosses. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; †††† p < 0.0001.

Figure 2

IFN-𝛼2 continues to be effective in decreasing TNBC cell viability despite a knockdown in SLFN12. Crystal violet assay showed a decrease in cell viability with IFN-α2 and a further decrease when AdvShSLFN12 and IFN-α2 were combined in (A) MDA-MB-231 (n = 6, p < 0.0001), (B) BT-549 (n = 6, p < 0.0001), and (C) Hs-578T (n = 4, p = 0.0001). All error bars shown represent standard error of the mean. Asterisks denote significance between control and each condition, whereas crosses indicate significance between shown conditions. p-value is for both asterisks and crosses. ** p < 0.01; *** p < 0.001; **** p < 0.0001; † p < 0.05; †† p < 0.01; †††† p < 0.0001.

Figure 3

SLFN family expression variably increases with loss of SLFN12 and IFN-𝛼2 signaling. mRNA analysis by primer-probe RT-qPCR showed that (A) SLFN5 (n = 6, p < 0.0001), (B) SLFN12-Like (n = 6, p < 0.0001), and (C) SLFN14 (n = 6, p < 0.0001) are induced by IFN-𝛼2 treatment and significantly further induced with the loss of SLFN12 in MDA-MB-231 cells. This relationship indicates further SLFN family interplay in expression regulation. Contrariwise, (D) SLFN11 (n = 6, p = 0.0001) and (E) SLFN13 (n = 6, p < 0.0001) increased in expression due to IFN-𝛼2 treatment, but this increase was lost with the knockdown of SLFN12 in MDA-MB-231 cells, indicating that SLFN12 may regulate SLFN11 and 13 expression during IFN-𝛼2 signaling. RPLP0 is used as reference gene. All error bars shown represent standard error of the mean. Asterisks denote significance between control and each condition, whereas crosses indicate significance between shown conditions. p-value is for both asterisks and crosses. *** p < 0.001; **** p < 0.0001; †† p < 0.01; ††† p < 0.001; †††† p < 0.0001.

Figure 4

SLFN11 and SLFN13 mRNA and protein expression increase with IFN-α2 signaling. MDA-MB-231 protein levels were analyzed by flow cytometry to measure (A) SLFN11 (n = 16, p < 0.0001), (B) SLFN12 (n = 16, p < 0.0001), and (C) SLFN13 (n = 14, p < 0.0001). All 3 SLFNs showed an increase in protein expression following IFN-α2 treatment alone compared to control and the combination of AdvShSLFN12 with IFN-α2 in comparison to AdvShSLFN12 alone. There was no significant change in protein expression with the loss of SLFN12 alone in SLFN11, SLFN12, or SLFN13. All error bars shown represent standard error of the mean. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 5

Apparent SLFN Interplay with IFN-α2 Signaling. mRNA analysis by primer-probe RT-qPCR in MDA-MB-231 cells evaluating the expression of (A) SLFN5 (n = 6, p < 0.0001), (C) SLFN12-Like (n = 6, p < 0.0001), (E) SLFN14 (n = 3, p < 0.0001), and (B,D,F) SLFN12 after respective siRNA treatments. Astricts denote significance between shown bars, and # denotes significance between AdvShSLFN12 + siNT and siSLFN5 + AdvShSLFN12 expression. p-value is for both asterisks and number signs. RPLP0 is used as reference gene. All error bars shown represent standard error of the mean. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ## p < 0.01; #### p < 0.0001.

Figure 6

IFN-α2 decreases TNBC cell viability with simultaneous knockdown of SLFN5, SLFN12, SLFN12-Like, and SLFN14. MDA-MB-231 cells were analyzed by flow cytometry with Zombie Aqua™ Fixable Viability Kit. (A) SLFN5, SLFN12, SLFN12-Like, and SLFN14 (n = 6, p < 0.0001) were individually knocked down, treated with IFN-α2 individually and in combination. Live cells are displayed as Zombie, and dead cells are represented by Zombie +. Two-way ANOVA analysis was performed. * denote significance to Scramble + siNT, † specifies significance to Scramble + siNT; # shows the comparison of Scramble + siNT + IFN-α2 vs. AdvshSlfn12/siRNAs + IFN-α2, and $ indicates the comparison of each AdvshSlfn12/siRNA vs. its respective AdvshSlfn12/siRNA + IFN-α2. All error bars shown represent standard error of the mean. (B) Treatments were then ranked by the largest decrease in cell viability following IFN-α2 treatment by the mean and SEM of the Zombie + cell values. *, †, #, $ p < 0.05.

Figure 7

Proposed IFN-α2 signaling cascade. IFN-α2 signaling cascade that illustrates a novel intra-regulation between Schlafen family members and increasing control of cell viability.