The ZNF304-integrin axis protects against anoikis in cancer

Abstract

Ovarian cancer (OC) is a highly metastatic disease, but no effective strategies to target this process are currently available. Here, an integrative computational analysis of the Cancer Genome Atlas OC data set and experimental validation identifies a zinc finger transcription factor ZNF304 associated with OC metastasis. High tumoral ZNF304 expression is associated with poor overall survival in OC patients. Through reverse phase protein array analysis, we demonstrate that ZNF304 promotes multiple proto-oncogenic pathways important for cell survival, migration and invasion. ZNF304 transcriptionally regulates β1 integrin, which subsequently regulates Src/focal adhesion kinase and paxillin and prevents anoikis. In vivo delivery of ZNF304 siRNA by a dual assembly nanoparticle leads to sustained gene silencing for 14 days, increased anoikis and reduced tumour growth in orthotopic mouse models of OC. Taken together, ZNF304 is a transcriptional regulator of β1 integrin, promotes cancer cell survival and protects against anoikis in OC.

Figure 1

Significance of zinc finger protein 304 (ZNF304) expression in human ovarian carcinoma (OC). Abbreviations: N-cad, N-cadherin; CNTFR, ciliary neurotrophic factor receptor; MAGED1, melanoma antigen family D, 1; NR2F2, nuclear receptor subfamily 2, group F, member 2. (A) Graphical representation of computational analysis using The Cancer Genome Atlas high-grade serous ovarian cancer (OC) dataset. (B–C) Kaplan-Meier curves for patients with OC on the basis of ZNF304 expression in (B) the training set and (C) the validation set. Kaplan-Meier curves indicate that high ZNF304 expression is a predictor of poor overall survival in patients with OC (n = 88, P = 0.03). (D) Western blot analysis of ZNF304 protein expression in 7 ovarian cell lines. (E) Reverse transcription polymerase chain reaction analysis of ZNF304 mRNA levels in 7 ovarian cell lines.

Figure 2. Silencing zinc finger protein 304 (ZNF304) inhibits tumor cell invasion, migration, and proliferation

(A) Invasion %, (B) migration % of HeyA8 cells, and (C) migration % of SKOV3IP1 cells. Migration and invasion percentages in ZNF304 siRNA treated samples were calculated after normalization with control siRNA treated samples. Data are presented as mean ± SEM. (D) Western blot analysis of p-Src (Y416) and total Src levels (E) focal adhesion kinase (FAK) phosphorylation in wild-type cells and cells in which ZNF304 had been knocked out by 1 of 3 siRNA constructs (F) β1 integrin and paxillin phosphorylation at tyrosine 31 and tyrosine 118 sites after 72 hours of ZNF304 siRNA treatment in HeyA8 cells (G–J) Cell-cycle arrest analysis of HeyA8 cells (G), SKOV3IP1 cells (H), A2780PAR cells (I), and A2780P20 cells (J) after 72 hours transfection with ZNF304 siRNA. Cells were harvested at 72 hours and were fixed, stained with propidium iodide, and analyzed by fluorescence-activated cell sorting. Data are presented as the percentage of cells (mean ± SEM).

Figure 3. Zinc finger protein 304 (ZNF304) associates with integrin beta 1 (ITGB1) promoter and regulates β1 integrin expression

(A) Western blot analysis of ZNF304 and β1 integrin protein expression (B) We identified 10 predicted binding sites of ZNF304 in the ITGB1 promoter on the basis of support vector machine scores using an online tool, which is available at http://compbio.cs.princeton.edu/zf/. (C), ITGB1 promoter with 10 predicted binding sites and 6 primer sets were designed for the 10 predicted binding sites. (D) Chromatin immunoprecipitation (ChIP) analyses with ZNF304 antibody in HeyA8 cells. Relevant sequences were quantified by polymerase chain reaction with 6 pre-designed primers subsequent to ChIP assay. (E) Densitometric analysis of ChIP analysis. Sequence and antibody specificity controls were included. Data are presented as percentage of input. (F) Luciferase activity after HeyA8 cells were treated with control siRNA (black) or ZNF304 siRNA (grey). Fold of induction was calculated after normalization with empty vector. Data are presented as means ± standard error of the mean (SEM). Luciferase activity was inhibited after control siRNA treatment or ZNF304 siRNA treatment in BS1-vector–transfected cells, in BS2-vector–transfected cells, and in BS-3-vector– transfected cells. (G) Luciferase activity increased after transfection of ZNF304-expressing vector into BS1-, BS2-, and BS3-vector–transfected HeyA8 cells. Data are presented as means ± SEM.

Figure 4. Zinc finger protein 304 (ZNF304)-mediated inside-out signalling

(A) The in vitro anoikis rates of HeyA8 cells in suspension conditions at 72 hours (B) Poly ADP ribose polymerase cleavage in ZNF304 siRNA-treated and control siRNA-treated samples in suspension conditions. (C) The anoikis rate of the HeyA8 and SKOV3IP1 cells in suspension condition after ZNF304 silencing and β1 integrin overexpression.

Figure 5. Sustained in vivo Zinc Finger Protein 304 (ZNF304) gene silencing

(A) Size and (B) zeta potential of dual assembly nanoparticles (DANPs) determined by Zeta Sizer (C) Atomic force microscopy images of DANP show the morphology and size distribution of particles. (D) Biodistribution of rhodamine 6G–labeled DANP in vivo. Tumors and the major organs were removed 24 hours after a single administration of rhodamine 6G–labeled DANP. The nanoparticles were monitored using fluorescent microscopy and representative images were taken at 10X (left) and 20X magnification (center). Number of nanoparticles was counted at 5 fields per slide (right). Data are presented as means ± standard error of the mean (SEM). (E) Sustained in vivo ZNF304 silencing in HeyA8 orthotopic model of OC. Tumors were removed and analyzed by immunoblotting at 3, 7, and 14 days after a single administration of ZNF304 siRNA-DANP (F) Effect of DANP, DANP-Control siRNA and DANP-ZNF304 siRNA on cytokine levels in plasma at 72 hours, after a single intravenous administration. Inflammatory Cytokine responses were assessed in the serum of C57 black mice. Mice were treated with single i.v. injections of DANP alone (n=6), DANP-Control siRNA (n=6), and DANP-ZNF304 siRNA (n=6) and no treatment (n=2) and serum was collected after 72h using cardiac puncture. A Luminex assay designed to detect 12 pro-inflammatory cytokines was used.

Figure 6. Effects of in vivo zinc finger protein 304 (ZNF304) gene silencing on tumor growth and vasculature

(P-values obtained with Student’s t-test; *P<0.05; **P<0.01; ***P<0.001; or ****P<0.0001; compared with control siRNA treated group; bars and error bars represent mean values and the corresponding SEM. (A) Effect of ZNF304 siRNA-dual assembly nanoparticles (DANP) treatment on tumor weight (left panel) and number of nodules (right panel) in the HeyA8 orthotopic murine model. (B) Knockdown of ZNF304 by ZNF304 siRNA-DANP and the effect of treatment on SKOV3 tumor weight (left) and number of nodules (right). (C) Immunohistochemical staining for tumor proliferation (Ki67) and microvessel density (CD31) in the SKOV3 orthotopic murine model of ovarian cancer. Quantification of Ki67 positive and CD31 positive cells in Control siRNA and ZNF304 siRNA treated groups are shown in Supplementary Figure 14. (D) Kaplan-Meier survival curve illustrating the effects of DANP-ZNF304 siRNA treatment versus Control siRNA treatment for the in vivo OVCA-432 survival model. Survival curves indicate that biweekly treatment of DANP-ZNF304 siRNA improves survival in vivo [n =8/group, P= 0.01 (Control siRNA versus ZNF304 siRNA), Log-rank (Mantel-Cox) test] (E) Viability of epithelial cells in ascites of mice. ZNF304 siRNA-DANP was administered intravenously when ascites was detectable. Ascites was removed seven days after a single administration and viability of epitelial cells were detected by FITC-Epcam and PI staining followed by flow cytometry. (n=3, P <0.0001).

Figure 7. Schematic representation

Schematic representation of mechanisms by which ZNF304 downregulation results in decreased cell growth and increased anoikis in tumor cells.