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. 2016 Jul 12;7(28):43689-43702.
doi: 10.18632/oncotarget.9905.

Blocking the PAH2 domain of Sin3A inhibits tumorigenesis and confers retinoid sensitivity in triple negative breast cancer

Nidhi Bansal  1 , Almudena Bosch  1 , Boris Leibovitch  1 , Lutecia Pereira  2 , Elena Cubedo  2 , Jianshi Yu  3 , Keely Pierzchalski  3 , Jace W Jones  3 , Melissa Fishel  1 , Maureen Kane  3 , Arthur Zelent  2 , Samuel Waxman  1 , Eduardo Farias  1
Affiliations

Blocking the PAH2 domain of Sin3A inhibits tumorigenesis and confers retinoid sensitivity in triple negative breast cancer

Nidhi Bansal et al. Oncotarget. .

Abstract

Triple negative breast cancer (TNBC) frequently relapses locally, regionally or as systemic metastases. Development of targeted therapy that offers significant survival benefit in TNBC is an unmet clinical need. We have previously reported that blocking interactions between PAH2 domain of chromatin regulator Sin3A and the Sin3 interaction domain (SID) containing proteins by SID decoys result in EMT reversal, and re-expression of genes associated with differentiation. Here we report a novel and therapeutically relevant combinatorial use of SID decoys. SID decoys activate RARα/β pathways that are enhanced in combination with RARα-selective agonist AM80 to induce morphogenesis and inhibit tumorsphere formation. These findings correlate with inhibition of mammary hyperplasia and a significant increase in tumor-free survival in MMTV-Myc oncomice treated with a small molecule mimetic of SID (C16). Further, in two well-established mouse TNBC models we show that treatment with C16-AM80 combination has marked anti-tumor effects, prevents lung metastases and seeding of tumor cells to bone marrow. This correlated to a remarkable 100% increase in disease-free survival with a possibility of "cure" in mice bearing a TNBC-like tumor. Targeting Sin3A by C16 alone or in combination with AM80 may thus be a promising adjuvant therapy for treating or preventing metastatic TNBC.

Keywords: SID decoys; Sin3A; metastases; retinoids; triple negative breast cancer.

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Conflict of interest statement

The authors have declared that no conflicts of interest exists.

Figures

Figure 1
Figure 1. SID peptide increases expression of RARβ2, endogenous retinoic acid levels and activates RARE-driven promoter
(A) qRT PCR for expression of RARβ2 in MDA-MB-231 cells treated with 2.5 μM SID peptide for 24 h, 72 h and 144 h. Error bars represent mean ± SD (n = 3). SCR vs SID, *p = 0.0110 (24 h); ***p < 0.0001 (72 h and 144 h), unpaired t-test. (B) Endogenous retinoic acid levels in MDA-MB-231 and 4T1 cells, untreated or treated with 2.5 μM SCR or SID peptides. Error bars represent mean ± SD (n = 3). SCR versus SID, *p = 0.0145 (MDA-MB-231); **p = 0.0051 (4T1); unpaired t-test. (C) Expression of RARE-driven GFP reporter in MDA-MB-231 variant D3H2LN, MDA-MB-157 and MMTV-Myc cells treated with 2.5 μM SCR or SID peptides. Error bars represent mean ± SD (n = 3). SCR vs SID, *p = 0.0219 (D3H2LN); **p = 0.004 (MDA-MB-157); ***p = 0.00031 (MMTV-Myc), unpaired t-test.
Figure 2
Figure 2. C16 modulates expression of RARs and enhances the retinoid signaling in combination with AM80
(A) qRT PCR for expression of RARα1/2, RARβ1/2 and RARγ1 in MDA-MB-231 cells treated with 200 nM C16 for 96 h. Error bars represent mean ± SD (n = 3). DMSO versus C16, ***p < 0.0001, unpaired t-test. (B) Ratio of relative expression of RARs measured in (A). (C) Western Blot for RARα, RARβ and RARγ1 in MDA-MB-231 cells treated with C16 at 200 nM for 96 h. (D) Endogenous retinoic acid levels in three breast cancer cell lines (MDA-MB-231, 4T1 and MMTV-Myc) treated with 200 nM C16 for 6 days. Error bars represent mean ± SD (n = 3). DMSO versus C16, **p = 0.0059 (MDA-MB-231); p = 0.0022 (4T1), p = 0.008 (MMTV-Myc), unpaired t-test. (E) Expression of RARE-driven GFP reporter in MDA-MB-231 cells treated with 200 nM C16 and/or 200 nM AM80 (n = 2). DMSO versus C16-AM80, *p < 0.05, one-way ANOVA.
Figure 3
Figure 3. C16 in combination with AM80 induces morphogenesis and inhibits tumorsphere formation
(A) Colony morphogenesis of 4T1 cells cultured in 3D Matrigel with 200 nM C16 and or 200 nM AM580 for 10 d followed by staining with DAPI (blue), caspase-3 (green) and phalloidin (red). Scale bar = 25 μm. Arrows indicate the partial acini formation. (B) Colony morphogenesis of 4T1 cells cultured in 3D matrigel with 200 nM C16 and/or 200 nM AM80 for 10 d followed by staining of the colonies with DAPI (blue) and caspase-3 (green). Scale bar = 50 μm. Arrows indicate the partial acini formation. (C) Tumorsphere assay of 4T1 cells treated with 200 nM C16 and/or 200 nM of AM80 for 7 d. Results show numbers of tumorspheres. Error bars represent mean ± SD (n = 3). DMSO versus C16 or AM80 or C16-AM80, ***p < 0.001, unpaired t-test. (D) Tumorsphere assay in MMTV-Myc cells treated with 200 nM C16 and/or 200 nM of AM80 for 7 d. Results show numbers of tumorspheres. Error bars represent mean ± SD (n = 3). DMSO versus C16, **p = 0.0064; DMSO versus C16-AM80, **p = 0.0019, unpaired t-test.
Figure 4
Figure 4. C16 prevents development of Myc-driven mammary hyperplasia
(A) Representative images of the mammary gland architecture of 30 weeks old virgin MMTV-Myc oncomice (n = 16/group) treated with DMSO or C16 for 20 weeks. The far right panel shows an age-matched control of mammary gland isolated from healthy virgin FVB mouse. (B) Graph showing the number of side branches per mm of the mammary glands (n = 32) isolated in (A). DMSO versus C16, ***p < 0.0001, one-way ANOVA. (C) Kaplan-Meier plot showing tumor-free survival of MMTV-Myc oncomice treated with DMSO or C16 (n = 8/group). DMSO versus C16, *p = 0.0272, p, logrank test.
Figure 5
Figure 5. C16 in combination with AM580 inhibits primary tumor growth and lung metastases
(A) Tumor progression in Balb/c mice (n = 8) inoculated with 4T1 cells and then treated with DMSO or C16 or AM580 alone or in combination for 17 days, and tumor volume quantified at the indicated times. DMSO versus C16, *p = 0.0273; DMSO versus C16-AM580, **p = 0.0032, unpaired t-test. (B) Lungs from sacrificed animals (A) were isolated and metastatic foci counted. DMSO vs C16-AM580, *p = 0.0158, Mann-Whitney test.
Figure 6
Figure 6. Adjuvant treatment with C16-AM80 combination inhibits metastatic dissemination and increases disease-free survival
(A) Kaplan-Meier analysis showing disease-free survival following removal of primary tumors in Balb/c mice (n = 5) inoculated with 4T1 cells and treated with C16 and AM80 alone or in combination. DMSO versus C16, *p = 0.0343; DMSO versus C16-AM80, **p = 0.0025, logrank test. (B) Lungs from sacrificed Balb/c mice inoculated as described in (A) were isolated and metastases counted. DMSO versus, C16, *p = 0.0159, Mann-Whitney test; DMSO versus AM80, *p = 0.0286, Mann-Whitney test; DMSO versus C16-AM80, **p = 0.0039, one-way ANOVA (Mann-Whitney could not be applied due to repeated values of zero for C16-AM80 treated mice). (C) Quantification of the disseminated 4T1 tumor cells isolated from the bone marrow of sacrificed animals from (A). DMSO versus, C16, **p < 0.01; DMSO versus C16-AM80, **p < 0.01, one-way ANOVA. (D) Kaplan-Meier analysis showing disease-free survival following removal of primary tumors in Balb/c mice inoculated with 4T1 cells and treated with C16 and atRA alone or in combination. DMSO versus C16, *p = 0.02345, logrank test. (E) Lungs from sacrificed Balb/c mice inoculated as described in (D) were isolated and quantified for the number of metastases observed. DMSO versus C16 or C16-atRA, *p = 0.0286, Mann-Whitney test. (F) Quantification of the disseminated 4T1 tumor cells isolated from the bone marrow of sacrificed animals from (D).
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