Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance

Abstract

Triple-negative breast cancer (TNBCs) account for 15-20% of all breast cancers and represent the most aggressive subtype of this malignancy. Early tumor relapse and progression are linked to the enrichment of a sub-fraction of cancer cells, termed breast tumor-initiating cells (BTICs), that undergo epithelial to mesenchymal transition (EMT) and typically exhibit a basal-like CD44^high/CD24^low and/or ALDH1^high phenotype with critical cancer stem-like features such as high self-renewal capacity and intrinsic (de novo) resistance to standard of care chemotherapy. One of the major mechanisms responsible for the intrinsic drug resistance of BTICs is their high ALDH1 activity leading to inhibition of chemotherapy-induced apoptosis. In this study, we demonstrated that aurora-A kinase (AURKA) is required to mediate TGF-β-induced expression of the SNAI1 gene, enrichment of ALDH1^high BTICs, self-renewal capacity, and chemoresistance in TNBC experimental models. Significantly, the combination of docetaxel (DTX) with dual TGF-β and AURKA pharmacologic targeting impaired tumor relapse and the emergence of distant metastasis. We also showed in unique chemoresistant TNBC cells isolated from patient-derived TNBC brain metastasis that dual TGF-β and AURKA pharmacologic targeting reversed cancer plasticity and enhanced the sensitivity of TNBC cells to DTX-based-chemotherapy. Taken together, these findings reveal for the first time the critical role of AURKA oncogenic signaling in mediating TGF-β-induced TNBC plasticity, chemoresistance, and tumor progression.

Fig. 1. AURKA expression is essential to mediate TGF-β-Induced ALDH activity.

a The Claudin low (CL)-TNBC subgroup (72 cases) showed an average age at diagnosis of 61.9 years, while the overall survival average was 126.5 months. The median months' survival calculated for the whole follow-up was 27.96 months. In total, 12/72 cases (17%) exhibited AURKA alterations (mRNA up-regulation and/or copy number variations), while none of 72 patients harbored deletions or down-regulations of AURKA. Survival analysis showed that aberrant AURKA expression was significantly associated with reduced patient overall survival (p-value = 0.00584). b Immunoblot assay showing expression of total and phosphorylated AURKA in BT-549 cells before and after treatment with TGF-β1 (10 ng/ml) for 48 h. c Densitometry analysis showing the fold change of total and phosphorylated AURKA protein levels in BT-549 TNBC cells normalized to α-Tubulin. Experiments were performed in triplicate. d BT-549 cells were treated with TGF-β1 (10 ng/ml) for 48 h. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as control. Graph showing the average of ALDH1^High cells from three independent experiments (±s.d.). e Immunoblot assay showing expression of total AURKA in BT-549 cells infected with scrambled and AURKA lenti-shRNAs for 48 h. Densitometry analysis showing the fold change of total AURKA protein levels in BT-549 TNBC cells normalized to α-Tubulin. f BT-549 cells were treated with 10 ng/ml TGF-β1 and lenti-shRNAs. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. Graph showing the average of ALDH1^High cells from three independent experiments (±s.d.). g BT-549 cells were treated with 10 ng/ml TGF-β1, scrambled lenti-shRNAs, and lenti-shRNAs targeting SMAD3. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. Graph showing the average of ALDH1^High cells from three independent experiments (±s.d.).

Fig. 2. ALDH1 activity mediates AURKA-induced self-renewal capacity.

a Immunoblot assay showing expression of TGβR1, TGβR2, SMAD3 (total and phosphorylated), and AURKA (total and phosphorylated) proteins in MDA-MB 231 and SUM149-PT cells. Densitometry analysis showing the fold change of protein levels in MDA-MB 231 and SUM149-PT cells normalized to α-Tubulin. b SUM149-PT cells were treated with 10 ng/ml TGF-β1, lenti-shRNAs, or alisertib. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. c Graph showing the average of ALDH1^high cells from three independent experiments (±s.d.). d SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 1000 SUM149-PT cells derived from tertiary MPS were then infected with scrambled or AURKA lenti-ShRNAs and were incubated for 8 days to monitor MPS growth. Graph showing the average of ALDH1^High cells from three independent experiments (±s.d.). e FACS sorting analysis was performed on SUM149-PT secondary MPS to separate ALDH1^high from ALDH1^low cells. 10,000 ALDH1^high and ALDH1^low cells were then cultured for 8 days under non-adherent conditions to form tertiary MPS. Cancer cells were labeled with 5 μM Cell Tracker Red CMTPX (Thermo Fisher Scientific, #C34552) for 1 h and the MPS area was measured using the NIH Image-J software. Graph showing the average of ALDH1^High and ALDH1^low MPS area from three independent experiments (+/− s.d.). f GFP-tagged SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 10,000 SUM149-PT cells derived from tertiary MPS were then treated with DMSO (control) or 1 μM A37 (ALDH inhibitor) for 8 days. MPS area was measured using the NIH Image-J software. Graph showing the average of MPS area from three independent experiments (±s.d.).

Fig. 3. AURKA genetic targeting restores chemosensitivity.

a BT-549 cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary mammospheres (MPS). In total, 10,000 cells derived from tertiary MPS were then infected with scrambled or AURKA lenti-shRNAs, treated with 10 ng/ml TGF-β1 in the presence of DMSO (control) or 10 nM docetaxel (DTX) and incubated for 8 days to monitor MPS growth. MPS were labeled with 5 μM Cell Tracker Red CMTPX (Thermo Fisher Scientific, #C34552) for 1 h and the MPS area was measured using the NIH Image-J software. b Graph showing the average of MPS area from three independent experiments (±s.d.). c GFP-tagged SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then infected with scrambled or AURKA lenti-shRNAs and treated with DMSO (control) or 10 nM DTX for 8 days to monitor MPS growth. MPS area was measured using the NIH Image-J software. d Graph showing the average of MPS area from three independent experiments (±s.d.).

Fig. 4. Transcriptomic analysis of MDA-MB 231 TNBC cells.

a RNA-Seq analysis was performed on MDA-MB 231 cells treated with 10 ng/ml TGF-β1 for 48 h. 14,239 genes were differentially expressed between control and TGF-β1 groups. b STRINGdb software was used to identify a SNAI1/MMP9/FN1 Network. c Quantification analysis showing FN1, MMP9, and SNAI1 expression before and after TGF-β1 treatment. Experiments were performed in triplicate with a p-value < 0.05. d Real-time quantitative RT-PCR to detect SNA1, FN1, and MMP9 gene expression using MDA-MB 231 cells treated with 10 ng/ml TGF-β1 and infected with scrambled Lenti-shRNA or Lenti-shRNA targeting AURKA (Origene) for 48 h. Three independent experiments were performed in triplicate (±S.D. and P-value < 0.05).

Fig. 5. SNAI1 genetic targeting in TNBC cells.

a BT-549 cells were treated with 10 ng/ml TGF-β1, scrambled lenti-shRNAs and lenti-shRNAs targeting SNAI1. After 48 h incubation, ALDH1 activity was detected with Aldefluor kit and measured by FACS analysis on 10,000 events. ALDH1 inhibitor DEAB was used as the control for each sample. Graph showing the average of ALDH1^High cells from three independent experiments (±s.d.). b SUM149-PT cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then infected with scrambled or SNAI1 lenti-shRNAs and treated with DMSO (control) or 10 nM DTX for 8 days to monitor MPS growth. MPS were labeled with 5 μM Cell Tracker Red CMTPX (Thermo Fisher Scientific, #C34552) for 1 h and MPS area was measured using the NIH Image-J software. c Graph showing the average of MPS area from three independent experiments (±s.d.). d In total, 12,000 cells infected with scrambled or AURKA and/or SNAI1 lenti-shRNAs were plated on 96-well costar plates and Real-time wound healing assay was performed by using the IncuCyte Instrument. Experiments were performed in triplicate (±s.d.). e In total, 50,000 TNBC-M40 cells infected with Luciferase lenti-vectors and scrambled Lenti-shRNA or SNAI1 scrambled Lenti-shRNA constructs were transplanted into the mammary fat pad of female NSG mice (three animals per group). Tumorigenic capacity was monitored in living animals at 0 (2 h post-injection was used as the control for cell viability) and 7 days post-injection by luciferase imaging. f The tumor Growth Area was quantified using ImageJ-NIH Software and represents the average of three animals per group.

Fig. 6. Dual pharmacologic targeting of TGF-β and AURKA oncogenic pathways.

a Immunoblot assay showing SNAIL protein expression in MDA-MB 231 cells infected with scrambled lenti-shRNAs or lenti-shRNAs targeting SNAI1 for 48 h. Densitometry analysis showing the fold change of SNAIL protein levels in MDA-MB 231 TNBC cells normalized to α-Tubulin. Experiments were performed in triplicate. b Immunoblot assay showing SNAIL and cleaved-PARP expression in MDA-MB 231 cells treated with galunisertib (50 nM) and/or alisertib (50 nM) for 48 h. Densitometry analysis showing the fold change of SNAIL and cleaved-PARP protein levels in MDA-MB 231 TNBC cells normalized to α-Tubulin. Experiments were performed in triplicate. c Establishment of MDA-MB 231 LM xenografts: 1 × 10⁶ cells were injected into the mammary fat pad of 4 weeks old female NSG mice. After 2 weeks of tumor growth, mice were randomized into six groups (five animals each group) and treated with 10 mg/Kg DTX (IP injections), 50 mg/Kg galunisertib (oral gavage), and 50 mg/Kg alisertib (oral gavage) 3 times/week for 3 weeks. After drug treatment, tumor relapse was monitored for additional 3 weeks or when the tumor xenografts reached a volume comparable to control groups. Tumor volume was measured 3 times/week using a digitalized caliper. d Following drug treatment and tumor relapse, mice were sacrificed and organ metastatic burden was determined ex-vivo using the Xenogen imaging system.

Fig. 7. Dual targeting of TGF-β and AURKA oncogenic pathways inhibits TNBC plasticity.

a Immunoblot analysis showing higher phospho-SMAD3 and phospho-AURKA in TNBC-M14 and TNBC-M25 tertiary MPS compared to MDA-MB 231 used as control. Graphs showing the densitometric quantification of total and phosphoproteins expression normalized to Tubulin. b GFP-tagged TNBC-M14 and TNBC-M25 cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then treated with DMSO (control) or DTX for 8 days to monitor MPS growth. MPS area was measured using the NIH Image-J software. c Graph showing the average of MPS area from three independent experiments (±s.d.). d TNBC-M14 and TNBC-M25 cells were treated with 50 nM galunisertib, 50 nM alisertib, and combination for 48 h. Expression and cellular localization of Vimentin (green) was assessed by Immunofluorescence employing the Zeiss Confocal Fluorescent Microscope. Experiments were performed in triplicate with similar results. e Adherent and mammospheres-derived TNBC-M14 and TNBC-M25 were treated with 50 nM galunisertib (Gala), 50 nM alisertib (Alis), and combination. After 48 h incubation, ALDH activity was measured by FACS analysis on 10,000 events. ALDH inhibitor DEAB was used as control. Experiments were performed in triplicate (±s.d.). f TNBC-M14 and TNBC-M25 cells were cultured under non-adherent conditions for 24 days (three serial passages) to form tertiary MPS. In total, 10,000 cells derived from tertiary MPS were then treated with 50 nM galunisertib, 50 nM alisertib, and combination for 8 days. MPS were labeled in red using the CellTracker Red CMTPX Dye. g MPS area was measured using the NIH Image-J software. Experiments were performed in triplicate (±S.D. and P-value < 0.005).

Fig. 8. Dual targeting of TGF-β and AURKA pathways enhances the sensitivity to docetaxel-based chemotherapy.

a, b Real-time apoptosis assay of TNBC-M14 and TNBC-M25 3D-MPS treated with 10 nM Docetaxel, 50 nM Galunisertib, and 50 nM Alisertib as single agents and in combination. Apoptotic cells were stained in red with ANNEXIN-V and quantified using the Cell Player System (IncuCyte, BioEssen). Experiments were performed in triplicate (±S.D. and P-value < 0.05). c Representative images of TNBC-M14 and TNBC-M25 3D-MPS treated with 10 nM Docetaxel, 50 nM Galunisertib, and 50 nM Alisertib as single agents and in combination. Apoptotic cells were stained in Red with ANNEXIN-V and quantified in real-time using the Cell Player System (IncuCyte, BioEssen). d TGF-β/AURKA oncogenic axis promotes the enrichment of chemoresistant ALDH^high BTICs: Bulk TNBC cells show a chemosensitive ALDH^low phenotype. Aberrant activation of TGF-β/AURKA/SNAI1 oncogenic axis induces TNBC plasticity resulting in the enrichment of ALDH1^high BTICs with intrinsic resistance to standard chemotherapeutic agents. Dual pharmacologic inhibition of TGF-β and AURKA pathways will impair TNBC plasticity and restore chemosensitivity through the selective targeting of ALDH1^high BTICs.