A mechanism linking Id2-TGFβ crosstalk to reversible adaptive plasticity in neuroblastoma

Abstract

The ability of high-risk neuroblastoma to survive unfavorable growth conditions and multimodal therapy has produced an elusive childhood cancer with remarkably poor prognosis. A novel phenomenon enabling neuroblastoma to survive selection pressure is its capacity for reversible adaptive plasticity. This plasticity allows cells to transition between highly proliferative anchorage dependent (AD) and slow growing, anoikis-resistant anchorage independent (AI) phenotypes. Both phenotypes are present in established mouse and human tumors. The differential gene expression profile of the two cellular phenotypes in the mouse Neuro2a cell line delineated pathways of proliferation in AD cells or tyrosine kinase activation/ apoptosis inhibition in AI cells. A 20 fold overexpression of inhibitor of differentiation 2 (Id2) was identified in AD cells while up-regulation of genes involved in anoikis resistance like PI3K/Akt, Erk, Bcl2 and integrins was observed in AI cells. Similarly, differential expression of Id2 and other genes of interest were also observed in the AD and AI phenotypes of human neuroblastoma cell lines, SK-N-SH and IMR-32; as well as in primary human tumor specimens. Forced down-regulation of Id2 in AD cells or overexpression in AI cells induced the cells to gain characteristics of the other phenotype. Id2 binds both TGFβ and Smad2/3 and appears critical for maintaining the proliferative phenotype at least partially through negative regulation of the TGFβ/Smad pathway. Simultaneously targeting the differential molecular pathways governing reversible adaptive plasticity resulted in 50% cure of microscopic disease and delayed tumor growth in established mouse neuroblastoma tumors. We present a mechanism that accounts for reversible adaptive plasticity and a molecular basis for combined targeted therapies in neuroblastoma.

Figure 1. Gene expression profiling of Neuro2a cells.

(A) Affymetrix microarray was performed with mRNA extracted from Neuro2a anchorage dependent (AD) and anchorage independent (AI) cells. 1180 differentially expressed genes (5% FDR, ≥1.5-fold change) were identified (see Table S1). In two-way hierarchical clustering analysis (clustering diagram), highly expressed genes were shown in red and weakly expressed genes in blue in AD and AI cell phenotypes (n = 4). (B) A representative Western blot analysis performed with protein extracted from Neuro2a AD and AI cells showed overexpression of n-myc and Id2 in the AD cells which correlated with the gene array results. (C) Representative Western blot analysis performed with protein extracted from Neuro2a AD and AI cells and densitometric band analysis revealed overexpression and activation of proteins involved in anoikis resistance in the AI phenotypes thereby validating the gene array profiling data. These include integrins, the anti-apoptotic protein Bcl2 and Akt, FAK/Src and Erk signaling molecules. The band intensity of the proteins was normalized to the intensity of GAPDH with the exception of phosphorylated proteins that were normalized to their total proteins. Data points represent mean ± S.D. (n = 4)

Figure 2. Differential protein profile in the AD and AI phenotypes of human neuroblastoma cells.

(A) A representative Western blot analysis performed with protein extracted from the AD and AI phenotypes of SK-N-SH and IMR-32 cells showed overexpression of n-myc and Id2 in the AD phenotype of both the cell lines. AI phenotypes of SK-N-SH cells show overexpression and activation of proteins involved in anoikis resistance including integrins and Akt, FAK/Src and Erk signaling molecules. For the IMR-32 cells, the phenotypic transition did not affect these pathways to the same extent. (B) Quantification of protein levels by densitometric band analysis (n = 3). The band intensity of the proteins was normalized to the intensity of GAPDH with the exception of phosphorylated proteins that were normalized to their total proteins. Data points represent mean ± S.D. (n = 3) (C) Immunofluorescence staining reveals the expression of Id2 protein in two human neuroblastoma specimens as well as in mouse tumor (Id2: green; nucleus: blue). The images were captured using 40x objective. Scale bar: 50 µm.

Figure 3. Id2 down regulation drives Neuro2a AD cells towards the AI phenotype.

(A, B) BrdU incorporation assay demonstrated that transfection of AD cells with Id2-siRNA reduced the rate of proliferation. The apoptotic cells were excluded by gating out the sub-2n cells. (C, D) AnnexinV staining revealed increased apoptosis in AD cells following transfection with Id2-siRNA. The percent apoptotic cells in the graph represent the sum of early and late apoptosis. (E, F) Cell cycle analysis showed reduced number of cells entering in S-phase after Id2 inhibition. (G) Western blot analysis validated the decreased expression of Id2 protein after Id2 inhibition in the AD cells. (H) Representative bands from Western blot analysis revealed over activation of Akt, Raf, Erk and Smad pathways and overexpression of Integrin β1 protein in AD cells after Id2 down regulation indicating activation of anoikis resistant pathways. Data points represent mean ± S.D. (n = 4–6). *p<0.04 and **p<0.0001 by Student’s t-test. Control or C: AD cells; siR or siRNA: AD cells transfected with siRNA against Id2; cR or cRNA: AD cells transfected with nonsense siRNA control; AS: AD cells transfected with Id2 antisense oligonucleotide; msm: AD cells transfected with mismatched oligonucleotide.

Figure 4. Effect of Id2 down regulation on human neuroblastoma cell lines.

(A) BrdU incorporation assay demonstrated that transfection of AD phenotype of SK-N-SH and IMR-32 cells with Id2-siRNA reduced the rate of proliferation. The apoptotic cells were excluded by gating out the sub-2n cells. (B) AnnexinV staining revealed increased apoptosis in AD phenotype of SK-N-SH and IMR-32 cells following transfection with Id2-siRNA. The percent apoptotic cells in the graphs represent the sum of early and late apoptosis. (C) Representative bands from Western blot analysis revealed over activation of Akt, Erk and Smad pathways and overexpression of Integrin β1 protein in both SK-N-SH and IMR-32 cells after Id2 down regulation. Data points represent mean ± S.D. (n = 4–6). *p<0.004 by Student’s t-test. Control or C: AD cells; siR or siRNA: AD cells transfected with siRNA against Id2; cR or cRNA: AD cells transfected with nonsense siRNA control.

Figure 5. Id2 overexpression increases cell proliferation in Neuro2a AI cells.

(A) Expression of green fluorescent protein (GFP) is evident in AI cells transfected with IRES-GFP or Id2-IRES-GFP plasmid. (B) Id2 protein is expressed in AI cells after Id2-IRES-GFP transfection, whereas Id2 protein was absent in AI cells transfected with IRES-GFP. (C) Representative plots showed 77–85% transfection efficiency. (D, E) BrdU incorporation assay indicating increased rate of proliferation in AI cells after Id2 overexpression. The apoptotic cells were excluded by gating out the sub-2n cells. (F) Representative bands from Western blot analysis revealed that overexpression of Id2 reduced the activation of Erk, Akt and Smad pathways in AI cells. Data points represent mean ± S.D. (n = 4–6). *p<0.04 by Student’s t-test. C: AI cells, Ires: AI cells transfected with IRES-GFP, Id2: AI cells transfected with Id2-IRES-GFP.

Figure 6. Stable knockdown of Id2 accelerates phenotypic transition.

(A) Western blot analysis reveals complete knockdown of Id2 on Neuro2a AD cells transduced with Id2-shRNA lentiviral particles. (B) After only 4 days in NeuroCult complete media, the Id2 knockdown cells readily formed dense large spheres while the scrambled-shRNA transduced cells and the non-transduced AD cells formed loose smaller spheres at the same time point suggesting that Id2 knockdown accelerates the transition from AD to AI phenotype in Neuro2a cells. The images were captured on an Olympus ckx41 microscope using 10x objective. (C, D) BrdU incorporation assay demonstrated that transduction of Neuro2a AD cells with Id2-shRNA lentivirus reduced the rate of proliferation. The apoptotic cells were excluded by gating out the sub-2n cells. Data points represent mean ± S.D. (n = 4). *p<0.006 by Student’s t-test. nt: nontransduced Neuro2a AD cells; sc: Neuro2a AD cells transduced with scrambled shRNA lentivirus; Id2⁻: Neuro2a AD cells transduced with Id2 shRNA lentivirus.

Figure 7. Id2 functions partially through TGFβ pathway.

(A, B) The TGFβ neutralizing antibody (1D11) and its I/II receptor inhibitor (LY2109761) induced apoptosis in the Id2-inhibited AD cells. Data points represent mean ± S.D. (n = 3). *p<0.0001 by Student’s t-test. (C) Representative bands from Western blot analysis revealed decreased Smad2/3 activation in the presence of both TGFβ inhibitors with no change in Akt, Raf, Erk or Bcl2 pathways when compared to the Id2-suppressed cells. (D) Co-immunoprecipitation study shows Id2 binding to both TGFβ and Smad2/3.

Figure 8. A model depicting the role of Id2 in Neuro2a phenotypic transition.

We show that the AI cells lose their proliferative potential due to loss of n-myc and Id2 expression when conditions induce loss of cell-matrix anchorage or serum starvation. The mechanism by which this occurs is through competitive binding of retinoblastoma (Rb) and TGFβ. Diminished Id2 enables Rb binding to E2F, thus blocking progression into the S-phase of cell cycling (free E2F induces S-phase genes) and inhibiting proliferation. Subsequently the cells either undergo apoptosis or develop resistance to anoikis. Concurrently, inhibiting Id2 enables TGFβ to activate the pathways of anoikis resistance allowing the cells to adapt to unfavorable conditions.

Figure 9. Treatment targeting AD and AI phenotypes as well as adaptive plasticity delayed mouse tumor growth.

(A) A graphical representation of the gross tumor presence demonstrates that the combined treatment with doxorubicin, metformin, sorafenib and LY2109761 cured 50% of mice with microscopic tumors. All treatments in (A) were started one day after neuro2a inoculation (n = 10–15). (B) Tumor volume curve during treatment of established tumors with a combination of doxorubicin, metformin, sorafenib and LY2109761 showed remarkably suppressed tumor growth (n = 10). All mice in the control group were sacrificed by day 11 after the tumors reached 20 mm in diameter. The drug treatment in (B) was started after 5 mm tumor was visible in mice. Dox: doxorubicin, met: metformin, soraf: sorafenib, LY: LY2109761.