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. 2022 Aug 31;11:e78163. doi: 10.7554/eLife.78163

Figure 6. Inhibition of WDR5 and mTOR cooperatively reduces translation and triple-negative breast cancer (TNBC) cell growth.

(A) Western blot analysis of the indicated proteins in LM2 cells treated for 3 days with or without 20 μM OICR-9429 in combination with control, 2 μM OSI-027, 2.5 μM temsirolimus, or 5 nM everolimus. (B) Western blot analysis of the indicated proteins in MDA-MB-453 treated for 3 days with or without 30 μM OICR-9429 in combination with control, 0.5 μM OSI-027, or 1 nM everolimus. (C) Normalized translational rates in LM2 cells from (A) (n=3, one sample t test). *p=0.0166 (DMSO versus OICR-9429), ***p=0.0001 (DMSO versus OSI-027), ** p=0.0099 (DMSO versus OICR-9429 +OSI-027), **p=0.0080 (DMSO versus everolimus), **p=0.0047 (DMSO versus OICR-9429+), *p=0.0232 (OSI-027 versus OICR-9429 +OSI-027), and *p=0.00352 (everolimus versus OICR-9429 +everolimus). (D) Normalized translational rates in MDA-MB-453 cells from (B) (n=3, one sample t test). *p=0.0107 (DMSO versus OICR-9429), ****p<0.0001 (DMSO versus OSI-027), ****p<0.0001 (DMSO versus OICR-9429 +OSI-027), **p=0.0012 (DMSO versus everolimus), ***p=0.0005 (DMSO versus OICR-9429 +everolimus), ****p<0.0001 (OSI-027 versus OICR-9429 +OSI-027), and *p=0.0370 (everolimus versus OICR-9429 +everolimus). (E–F) Colony formation assay of LM2 treated for 8 days with or without 20 μM OICR-9429 in combination with control or 2 μM OSI-027. Representative images (E) and quantification (F) are shown. ****p<0.0001 (DMSO versus OICR-9429), ****p<0.0001 (DMSO versus OSI-027), ****p<0.0001 (DMSO versus OICR-9429 +OSI-027), ****p<0.0001 (OICR-9429 versus OICR-9429 +OSI-027), and *p=0.0233 (OSI-027 versus OICR-9429 +OSI-027). (G–H) Colony formation assay of LM2 cells treated for 8 days with or without 20 μM OICR-9429 in combination with control or 5 nM everolimus. Representative images (G) and quantification (H) are shown. **p=0.0043 (DMSO versus OICR-9429), **p=0.0011 (DMSO versus everolimus), ***p=0.0004 (DMSO versus OICR-9429 +everolimus), **p=0.0042 (OICR-9429 versus OICR-9429 +everolimus), and *p=0.0272 (everolimus versus OICR-9429 +everolimus). (I–J) Colony formation assay of MDA-MB-453 treated for 10 days with or without 30 μM OICR-9429 in combination with control or 0.5 μM OSI-027. Representative images (I) and quantification (J) are shown. **p=0.0018 (DMSO versus OICR-9429), ***p=0.0009 (DMSO versus OSI-027), ****p<0.0001 (DMSO versus OICR-9429 +OSI-027), ****p<0.0001 (OICR-9429 versus OICR-9429 +OSI-027), and *p=0.011 (OSI-027 versus OICR-9429 +OSI-027). (K–L) Colony formation assay of MDA-MB-453 cells treated for 10 days with or without 30 μM OICR-9429 in combination with control or 1 nM everolimus. Representative images (K) and quantification (L) are shown (n=3, unpaired two-side Student’s t test). ***p=0.0002 (DMSO versus OICR-9429), ***p=0.0002 (DMSO versus everolimus), ****p<0.0001 (DMSO versus OICR-9429 +everolimus), ****p<0.0001 (OICR-9429 versus OICR-9429 +everolimus), and ***p=0.0003 (everolimus versus OICR-9429 +everolimus). Calculation of coefficients of drug interaction (CDIs) is described in Materials and methods section. Significant synergy is labeled with (#). For gel source data, see Figure 6—source data 1 and Figure 6—source data 2.

Figure 6—source data 1. Original western blots for Figure 6A.
Figure 6—source data 2. Original western blots for Figure 6B.

Figure 6.

Figure 6—figure supplement 1. Inhibition of WDR5 and mTOR cooperatively reduces translation and triple-negative breast cancer (TNBC) growth.

Figure 6—figure supplement 1.

(A) RT-quantitative PCR (qPCR) of selected differentially expressed genes (DEGs) in LM2 after DMSO, 2 μM OSI-027, 20 μM OICR-9429, or combined treatment for 3 days. (B) RT-qPCR of selected DEGs in MDA-MB-453 after DMSO, 0.5 μM OSI-027, 30 μM OICR-9429, or combined treatment for 3 days. (C) RT-qPCR of selected DEGs in LM2 after DMSO or 5 nM everolimus treatment for 3 days. (D) RT-qPCR of selected DEGs in MDA-MB-453 after DMSO or 1 nM everolimus treatment for 3 days. Significance determined by comparing each treatment to DMSO control (n=4, unpaired two-side Student’s t test). (E) Western blot analysis of the indicated proteins in LM2 shCtrl, shWDR5-1, and shWDR5-2 with or without hairpin induction by doxycycline (DOX) (1 μg/mL) for 3 days. (F) Western blot analysis of indicated proteins in LM2 shWDR5-1 with or without DOX (1 μg/mL) induction in combination with 3 days of control, 2 μM OSI-027, or 5 nM everolimus treatment. (G) Normalized translational rates of LM2 shWDR5 cells from (A) following 3 days of control, 2 μM OSI-027, or 5 nM everolimus treatment (n=3, one sample t test). (H–I) Colony formation assay of LM2 shWDR5 with or without hairpin induction by DOX in combination with control or 2 μM OSI-027 treatment for 8 days. Representative images (H) and quantification (I) are shown. (J–K) Colony formation assay of LM2 shWDR5 cells with or without hairpin induction by DOX in combination with control or 5 nM everolimus treatment for 8 days. Representative images (J) and quantification (K) are shown. (L–M) Colony formation assay of LM2 with or without 20 μM OICR-9429 in combination with control or 2.5 μM temsirolimus treatment for 8 days. Representative images (M) and quantification (L) are shown (n=3, unpaired two-side Student’s t test). *p<0.05; **p<0.001; ***p<0.001; ****p<0.0001. Calculation of coefficients of drug interaction (CDIs) is described in Materials and methods section. Significant synergy is labeled with (#). For gel source data, see Figure 6—figure supplement 1—source data 1 and Figure 6—figure supplement 1—source data 2.
Figure 6—figure supplement 1—source data 1. Original western blots for Figure 2—figure supplement 1E.
Figure 6—figure supplement 1—source data 2. Original western blots for Figure 2—figure supplement 1F.