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. 2012 Oct 1;72(19):5048-59.
doi: 10.1158/0008-5472.CAN-12-1248. Epub 2012 Aug 8.

Hedgehog signaling is a novel therapeutic target in tamoxifen-resistant breast cancer aberrantly activated by PI3K/AKT pathway

Bhuvaneswari Ramaswamy #  1   2 , Yuanzhi Lu #  3 , Kun-Yu Teng  3 , Gerard Nuovo  4 , Xiaobai Li  5 , Charles L Shapiro  1   2 , Sarmila Majumder  3   2
Affiliations

Hedgehog signaling is a novel therapeutic target in tamoxifen-resistant breast cancer aberrantly activated by PI3K/AKT pathway

Bhuvaneswari Ramaswamy et al. Cancer Res. .

Abstract

Endocrine resistance is a major challenge in the management of estrogen receptor (ER)-positive breast cancers. Although multiple mechanisms leading to endocrine resistance have been proposed, the poor outcome of patients developing resistance to endocrine therapy warrants additional studies. Here we show that noncanonical Hedgehog (Hh) signaling is an alternative growth promoting mechanism that is activated in tamoxifen-resistant tumors. Importantly, phosphoinositide 3-kinase inhibitor/protein kinase B (PI3K/AKT) pathway plays a key role in regulating Hh signaling by protecting key components of this pathway from proteasomal degradation. The levels of Hh-signaling molecules SMO and GLI1 and the targets were significantly elevated in tamoxifen-resistant MCF-7 cells and T47D cells. Serial passage of the resistant cells in mice resulted in aggressive tumors that metastasized to distant organs with concurrent increases in Hh marker expression and epithelial mesenchymal transition. RNAi-mediated depletion of SMO or GLI1 in the resistant cells resulted in reduced proliferation, clonogenic survival and delayed G(1)-S transition. Notably, treatment of resistant cells with PI3K inhibitors decreased SMO and GLI1 protein levels and activity that was rescued upon blocking GSK3β and proteasomal degradation. Furthermore, treatment of tamoxifen-resistant xenografts with anti-Hh compound GDC-0449 blocked tumor growth in mice. Importantly, high GLI1 expression correlated inversely with disease-free and overall survival in a cohort of 315 patients with breast cancer. In summary, our results describe a signaling event linking PI3K/AKT pathway with Hh signaling that promotes tamoxifen resistance. Targeting Hh pathway alone or in combination with PI3K/AKT pathway could therefore be a novel therapeutic option in treating endocrine-resistant breast cancer.

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Figures

Figure 1
Figure 1
Hh pathway is activated in tamoxifen-resistant breast cancer cells. A and B, real-time RT-PCR (A) and Western blot analysis of SHH, SMO, GLI1, and PTCH in MCF7 and OHTR cells (T100: OHTR100, T500: OHTR500, T1000: OHTR1000; B) quantified in the bar diagram. C, Western analysis and quantification of Hh markers in ER-positive cell lines. D and E, GLI-driven luciferase expression in MCF7 and OHTR cells (D) and after depletion of GLI1 in OHTR1000 cells (E). F and G, real-time RT-PCR (F) and Western analysis (G) of Hh target genes in MCF7 and OHTR cells quantified in the bar diagram.
Figure 2
Figure 2
Serial passage of tamoxifen-resistant xenografts in mice resulted in metastasis. A, growth curve of OHTR100 cells grafted in Balb/c nude mice (n = 5) at initial transplant (OHTR100), 2nd (OHTR2nd), and 3rd passage (OHTR3rd). B, H&E stain of lung and liver sections of mice-bearing OHTR3rd primary tumors. C, morphological changes acquired by OHTR100 cells upon serial passage in mice. D and E, Western analysis of E-cadherin and vimentin (D), Hh signaling and target proteins (E) in OHTR cells quantified in the bar diagram. F, immunocytochemical analysis of SMO, GLI, and BMI1, with insets at higher magnification.
Figure 3
Figure 3
SMO and GLI1 depletion inhibits tamoxifen-resistant cell growth. A, Western analysis of SMO and GLI1 knocked down OHTR2nd cells quantified in the bar diagram. B and C, cell proliferation assay (B) and clonogenic survival of SMO and GLI1 depleted cells in the absence (T0) and presence of 2 μmol/L tamoxifen (T2) quantified in the bar diagram (C). D, cell-cycle analysis of GLI1 depleted OHTR2nd cells without (T0) and with (T5) 5 μmol/L tamoxifen treatment. E and F, Western analysis of MYC (E) and real-time RT-PCR of MYC, SNAIL, and BMI1 (F) in SMO-depleted OHTR2nd cells.
Figure 4
Figure 4
PI3K/AKT pathway activates Hh signaling in tamoxifen-resistant cells. A, GLI- driven luciferase activity in OHTR2nd and MCF-7 cells treated with SHH. B, luciferase activity in OHTR2nd and MCF-7 cells treated with DMSO (Con) and 20 μmol/L LY294002 for 3 hours. C and D, luciferase activity in OHTR2nd cells overexpressing constitutively active P110 subunit of PI3K (P110*; C), and constitutively active AKT (AKT*) or the corresponding empty vectors (D), treated with 20 μmol/L LY294002 (LY). E and F, luciferase activity in OHTR2nd cells overexpressing dominant negative mutants of PI3K [P110(KR)] or AKT[AKT(KR); E] and in MCF-7 cells overexpressing P110* or AKT* (F). Ectopic expression of P110 and AKT mutants are shown in the inset. Western analysis of SMO and GLI1 in OHTR2nd cells treated with 20 μmol/L LY294002 for 3 hours (G), 50 mmol/L LiCl for 6 hours alone or in combination of 50 μmol/L MG132 for the last 1 hour (H), and of SMO in OHTR2nd cells treated with 20 μmol/L LY294002 (LY) and/or 50 mmol/L LiCl for 6 hours, or 50 μmol/L MG132 for the last 1 hour (I), as indicated. Bar diagrams present quantified data.
Figure 5
Figure 5
GDC-0449 inhibits growth of OHTR cells in vitro and in vivo. A, timeline of tumor induction and GDC-0449/tamoxifen treatment in mice. B, comparison of growth curve of OHTR2nd cell-induced xenografts in mice treated with GDC-0449 alone or in combination with tamoxifen (n = 8). Average tumor volume ± SEM is plotted against time (in days). C, representative images of tumor bearing mice. D and E, growth of OHTR2nd (D) and T47D (E) cells in the presence of GDC-0449 (50 μmol/L, G50) alone or in combination with tamoxifen (0.5 μmol/L, T0.5).
Figure 6
Figure 6
GLI1 expression inversely correlates with DFS and OS in primary human breast tumors. A, representative pictures of breast tumors (H&E), GLI1, and BMI1 (immunohistochemistry). B, comparison of DFS amongst breast cancer patients with ER-positive tumors expressing high versus low GLI1. C, comparison of DFS amongst breast cancer patients with node positive, ER-positive tumors expressing high versus low GLI1. D, comparison of overall survival amongst patients with breast cancer tumors expressing high versus low GLI1.
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