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. Author manuscript; available in PMC: 2016 Apr 13.
Published in final edited form as: Nat Commun. 2015 Jun 17;6:7351. doi: 10.1038/ncomms8351

The ZNF304-integrin axis protects against anoikis

Burcu Aslan 2,4, Paloma Monroig 2,10, Ming-Chuan Hsu 5,11, Guillermo Armaiz Pena 3, Cristian Rodriguez-Aguayo 2,4, Vianey Gonzalez-Villasana 2, Rajesha Rupaimoole 3, Archana Sidalaghatta Nagaraja 1,3, Selanere Mangala 3,4, Hee-Dong Han 14, Erkan Yuca 7, Sherry Y Wu 3, Cristina Ivan 3,4, Tyler J Moss 8, Prahlad T Ram 8, Huamin Wang 9, Alexandra Gol-Chambers 2, Ozgur Ozkayar 2,12, Pinar Kanlikilicer 2, Enrique Fuentes-Mattei 9, Nermin Kahraman 2, Bulent Ozpolat 2, Susan Tucker 6, Mien-Chie Hung 5,13, Keith Baggerly 6, Geoffrey Bartholomeusz 2, George Calin 1,2,4, Anil K Sood 1,3,4, Gabriel Lopez-Berestein 1,2,4,*
PMCID: PMC4830335  NIHMSID: NIHMS773901  PMID: 26081979

Abstract

Ovarian cancer is a highly metastatic disease, but no effective strategies to target this metastatic process currently are known. Here, an integrative computational analysis of The Cancer Genome Atlas ovarian cancer dataset coupled with experimental validation identified a novel zinc finger transcription factor ZNF304 as one of the key factors for ovarian cancer metastasis. High tumoral ZNF304 expression was associated with poor overall survival in ovarian cancer patients. Through reverse phase protein array analysis, we demonstrated 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 novel dual assembly nanoparticle led to sustained gene silencing for 14 days, increased anoikis, and reduced tumor growth in orthotopic mouse models of ovarian cancer. Taken together, ZNF304 is a novel transcriptional regulator of β1 integrin, promotes cancer cell survival, and protects against anoikis in ovarian cancer.

Introduction

Ovarian carcinoma (OC) has the highest mortality rate among gynecologic malignancies. In the United States in 2014, over 21,000 women will be diagnosed with OC, and more than 14,000 women will die 1. The most common histological subtype is high-grade serous OC (HGSOC), and the poor survival rate associated with this subtype is due primarily to the advanced stage of disease and widespread metastases at the time of diagnosis. The rapid spread of HGSOC is based on its propensity to seed the peritoneal cavity, leading to ascites formation and metastases 2,3; this highlights the need for a deeper understanding of the molecular mechanisms that regulate OC growth and progression. To identify new therapeutic targets and strategies, we carried out an integrative analysis of The Cancer Genome Atlas (TCGA) HGSOC dataset and gene profiles of ovarian and breast tumors to identify genes that are important for cancer metastasis. Among the genes identified, zinc finger protein 304 (ZNF304) was found to be the most highly associated with overall survival in HGSOC patients. ZNF304 is a transcription factor that belongs to the C2H2 zinc finger family. The member genes of this family represent the largest class of transcription factors in humans and, indeed, one of the largest gene families in mammals 4. ZNF304 can be upregulated by activated Kirsten rat sarcoma viral oncogene homolog (KRAS) in KRAS-positive colorectal cancer cells and binds at the promoters of INK4-ARF and other CpG island methylator phenotype genes in colorectal cancer cells and in human embryonic stem cells 5. However, the role of ZNF304 in metastasis and its downstream effectors are not well understood.

Here, we aimed to unravel the mechanisms by which ZNF304 promotes cancer metastasis and to evaluate its role as a potential therapeutic target.

Results

ZNF304 in human HGSOC

We first carried out an integrative computational analysis to identify genes that are important for cancer metastasis and that are upregulated in ovarian cancer (OC). Since N-cadherin has been reported to play a critical role in invasion and anoikis resistance of cancer cells 6,7, we first identified gene signatures in tumors with high N-cadherin expression in TCGA HGSOC dataset. Of 16,869 genes that were upregulated in OC, 493 genes had a positive correlation with tumoral N-cadherin expression (Figure 1A). Of these 493 genes, ciliary neurotrophic factor receptor (CNTFR); melanoma antigen family D, 1 (MAGED1); nuclear receptor subfamily 2, group F, member 2 (NR2F2), and ZNF304 were upregulated in invasive ovarian and breast tumor epithelium compared with normal ovarian 8and breast epithelium9, respectively.

Figure 1.

Figure 1

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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.

We then assessed the effect of tumoral expression on patient survival for these 4 genes using TCGA HGSOC dataset (Supplementary Figure 1). For each gene, we randomly split the entire OC patient population into training (2/3 of cases) and validation cohorts (1/3 of cases). In both cohorts, patients were divided into sextiles according to mRNA expression, and the first and last sextiles were contrasted. Importantly, the relationships between overall survival and known prognostic factors such as age or residual disease were examined in both the training and the validation cohorts using a Cox proportional hazards model. Only ZNF304 was a significant factor in this analysis (Figure 1B and 1C). In contrast, CNTFR (Training and validation sets; Supplementary Figure 1A and 1B, respectively), MAGED1 (Training and validation sets; Supplementary Figure 1C and 1D, respectively), and NR2F2 (Training and validation sets; Supplementary Figure 1E and 1F, respectively) expression levels were not correlated with patient survival. Patients with high tumoral ZNF304 expression had significantly lower median overall survival than patients with low tumoral ZNF304 expression (Figure 1B [training set, 22.2 versus 48.7 months, P = 0.031]; and Figure 1C [validation set, 40.4 versus 26.9 months, P = 0.039]). Based on these results, ZNF304 was selected for additional studies.

Next, we examined protein expression levels of ZNF304 (75 kDa) by Western blot analysis in 6 OC tumor cell lines and in HIO180 non-transformed ovarian epithelial cells (Figure 1D). ZNF304 protein was highly expressed in all OC cells tested, but a lower expression was observed in the HIO180 cells. ZNF304 mRNA basal levels were high in 4 of the 6 OC cell lines (Figure 1E). Additionally, we analyzed ZNF304 mRNA levels in patient normal distal fallopian tubes versus HIO180 and SKOV3IP1 cancer cells (Supplementary Figure 2). ZNF304 mRNA levels were similar between fallopian tube and HIO180; however, ZNF304 levels were significantly higher in SKOV3IP1 cells.

Downregulation of ZNF304 inhibits invasion, migration, and proliferation of OC cells

Given the potential role of ZNF304 in cancer metastasis, we next investigated whether silencing this target would affect invasion and migration. We first tested the knockdown efficiency of 3 siRNA sequences of ZNF304 in HeyA8 cells (Supplementary Figure 3). Two of the 3 siRNA sequences tested (ZNF304 siRNA-1 and ZNF304 siRNA-3) showed more than 65% inhibition of ZNF304 in HeyA8 cells. Therefore, these 2 siRNA sequences were selected for further studies. Next, we performed invasion and migration assays in HeyA8 and SKOV3IP1 cell lines with the selected siRNA sequences, resulting in 40% inhibition of invasion and 45% inhibition of migration in HeyA8 cells (Figure 2, A and B, respectively) and 27% inhibition of migration in SKOV3IP1 cells (Figure 2C).

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

Figure 2

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(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).

To determine the potential signaling pathways in which ZNF304 is involved, we performed a reverse phase protein array (RPPA) analysis of control siRNA-treated and ZNF304 siRNA-treated HeyA8 cells. Samples were probed with 214 validated antibodies to total proteins and the respective phospho-proteins. Silencing ZNF304 led to reduced expression of caveolin-1, fibronectin, MYH9 (myosin II), and the effectors of the Ras signaling pathway (BRAF, RAF1) (Supplementary Figure 4). This last finding guided us to further analyze the link between ZNF304 and integrin signaling (Supplementary Table 1). ZNF304 mRNA expression was highly correlated with β1 integrin expression in HGSOC samples (r = 0.20, P = 0.0015; Supplementary Figure 5).

To further understand the role of ZNF304, we validated the RPPA data and determined the levels of focal adhesion complex members after ZNF304 siRNA transfection in HeyA8 cells. Silencing ZNF304 decreased phosphorylation of Src and FAK, which are adaptor proteins of focal adhesion and major markers of cell migration (Figure 2, D and E, respectively). To further analyze pathways related to migration and invasion, we investigated the effects of ZNF304 silencing on paxillin and β1 integrin (Figure 2F). ZNF304 silencing inhibited both paxillin phosphorylation at tyrosine sites 31 and 118 and β1 integrin expression in the cell lines tested.

In the RPPA results, forkhead box M1 (FOXM1) and cyclin B1 levels were also decreased in ZNF304-silenced samples, suggesting that ZNF304 might play a role in the cell cycle. To determine the effects of ZNF304 silencing on proliferation, we performed cell-cycle analysis in HeyA8, SKOV3IP1, A2780PAR, and A2780CP20 cell lines after 72 hours of ZNF304 siRNA transfection (Figure 2, G–J, respectively). All cell lines treated with ZNF304 siRNA showed significant arrest in the G2 phase, confirming the decreases in cyclin B1 and FOXM1 levels found in the RPPA analysis.

ZNF304 transcriptionally regulates β1 integrin

The ZNF304 gene is located at chromosome 19q13.43 (www.genome.ucsc.edu). The ZNF304 protein consists of a Kruppel-associated box domain and 16 zinc finger proteins (UniProt) (Supplementary Figure 6). To explore the mechanism by which ZNF304 silencing downregulates migration, we determined protein and mRNA levels of β1 integrin upon ZNF304 siRNA treatment in HeyA8 and SKOV3IP1 cells. We first performed RT-PCR to determine the effects of the two selected ZNF304 siRNA sequences on β1 integrin mRNA levels; the results showed the downregulation of β1 integrin mRNA in these cells (Supplementary Figure 7A). We also determined reduced β1 integrin mRNA levels after ZNF304 silencing using real time RT-PCR (Supplementary Figure 7B–7D) .We determined the basal protein levels of β1 integrin and observed that it was expressed in the cell lines tested (Figure 3A).

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

Figure 3

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(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.

We next investigated whether ZNF304 transcriptionally regulates β1 integrin. ZNF304-DNA binding sites were predicted on the basis of support vector machines 10. We identified ten possible ZNF304 binding sites in the β1 integrin promoter by using support vector machine scores that ranged from 24.25 to 18.9 (Supplementary Figure 8). The transcription start site was predicted by the ensemble and was compared with the β1 integrin transcript sequence and the binding locations in the β1 integrin promoter region (Figure 3B). Six primer sets containing the segments for the 10 binding sites were designed (Figure 3C). DNA segments were amplified, cloned, sequenced, and confirmed with a standard nucleotide-nucleotide basic local alignment search tool (National Center for Biotechnology Information). To determine whether ZNF304 binds to the β1 integrin promoter, we performed chromatin immunoprecipitation assays (ChIP) in HeyA8 cells with ZNF304 antibody. Subsequent polymerase chain reaction (PCR) results confirmed the interaction of β1 integrin promoter and 5 of the 6 predicted ZNF304 binding sites (BS1, BS2, BS3, BS5, and BS6) (Figure 3D). A densitometric analysis of the inputs and immunoprecipitation results for each binding site revealed that BS1, BS2, and BS3 had an affinity of > 50% (Figure 3E). Owing to their affinity, BS1, BS2, and BS3 were selected for further studies.

To identify the role of ZNF304 in the regulation of β1 integrin gene transcription, we developed 3 constructs that each contained 1 of the binding sites and inserted them into pGL3-basic vector. HeyA8 cells were transfected with the constructs, and the activity of each binding site was determined by a dual-luciferase reporter assay in cells with basal ZNF304 expression and in cells in which ZNF304 had been knocked down by siRNA. As shown in Figure 3F, overall luciferase activity increased in cells transfected with the binding site constructs compared with cells transfected with the empty vector. Cells transfected with BS1-vector had 2 times more luciferase expression than empty vector cells, whereas BS2-vector–transfected cells had 6 times more luciferase expression than did the empty vector cells. Cells transfected with BS3-vector showed the highest luciferase activity (approximately 70 times more expression than empty vector cells). ZNF304 silencing led to a decrease in luciferase activity in all 3 binding sites. The most significant was BS2-transfected cells, which had a 40.3% inhibition of luciferase activity (P = 0.02; Figure 3F). We found a 13.8% decrease in luciferase activity for cells transfected with BS1-vector and a 7.8% decrease for cells transfected with BS3-vector, compared with control cells (P = 0.0173 and P = 0.2630, respectively). Co-transfection of ZNF304-expressing vector significantly induced the luciferase activity of BS1-, BS2-, and BS3-vector–transfected cells (Figure 3G). These results indicate that ZNF304 is a positive regulator of the active β1 integrin promoter and that ZNF304 increases its transcription by binding to BS2.

ZNF304 protects tumor cells from anoikis

β1 integrin confers a survival advantage to tumor cells 11. As a regulator of β1 integrin, ZNF304 can also inhibit anoikis through β1 integrin downregulation. Therefore, we examined the anoikis rates at 24, 48 and 72 hours in detached HeyA8 cells in vitro using polyhydroxyethylmethacrylate (poly-HEMA)-coated tissue culture plates that promote anchorage-independent cell growth 12. The anoikis rates in ZNF304 siRNA-transfected cells were significantly higher than Control siRNA-transfected cells at each time point tested (at 24, 48, and 72 hours) (Supplementary Figure 9). The highest anoikis rate and induction of PARP cleavage were observed at 72 hours; therefore all subsequent experiments were conducted at this time point. Cells transfected with the ZNF304 siRNAs for 72 hours had a significantly higher ([ZNF304 siRNA-1, P <0.0001]; [ZNF304 siRNA-3, P <0.0005]) rate of anoikis (75%-80%), than control untreated or control siRNA-treated cells (60%) (Figure 4A). Consistent with these results, immunoblotting from these samples showed that silencing ZNF304 also increased poly ADP ribose polymerase (PARP) cleavage (Figure 4B), which supports our observation of increased anoikis in cells transfected with ZNF304 siRNA.

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

Figure 4

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(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.

To determine the link between ZNF304-mediated β1 integrin and anoikis, we also performed a rescue experiment. HeyA8 and SKOV3IP1 cells were transfected with either control siRNA or ZNF304 siRNA. Next, cells were transiently transfected with either empty vector or β1 integrin–expressing vector and transferred to anoikis plates. Both HeyA8 and SKOV3IP1 cells that were transfected with β1 integrin–expressing vector showed increased survival and decreased anoikis rates (Figure 4C left and right, respectively). Furthermore, silencing ZNF304 increased the anoikis sensitivity and death rate of HeyA8 cells even in the presence of high β1 integrin expression. In addition, we generated mutations (5bp deletion on the binding site) on the ZNF304 overexpressing vector that led to insensitivity for ZNF304 siRNA-1 (Supplementary figure 10). We performed the rescue experiment using the ZNF304 overexpressing vector and mutant ZNF304 overexpressing vector. Immunoblotting showed that overexpression of mutant ZNF304 (insensitive for ZNF304 siRNA-1) led to a decrease in PARP cleavage, indicating the lack of anoikis in these cells (Supplementary Figure 10).

Sustained in vivo ZNF304 gene silencing

On the basis of our in vitro findings, we next investigated whether ZNF304 gene silencing would be effective in treating orthotopic murine models of OC. For the in vivo experiments, we developed and characterized a novel delivery system designed for sustained and prolonged gene silencing. Dual assembly nanoparticles (DANP) were prepared by using a chitosan core coated with polylactic acid (PLA). These particles had a diameter of 150–200 nm and a zeta potential of −10 mV, which corresponded to a neutral range (Figure 5, A and B, respectively). Atomic force microscopy (AFM) images demonstrated the spherical morphology and size distribution of the DANP (Figure 5C). This optimized nanoparticle formulation was used for all subsequent experiments owing to their small size, slight negative charge, and high efficiency at incorporating siRNA. We incorporated siRNA in the chitosan core by using chitosan/tripolyphosphate at a 3:1 ratio, which yielded more than 75% loading efficiency, as previously described 13. We next determined the tissue distribution of the DANP by labeling the particles with rhodamine 6G and administering these red fluorescence–labeled particles as a single dose intravenously to HeyA8 tumor–bearing mice. Twenty-four hours later, the mice were euthanized, and their major organs and the tumors were removed, processed, and sectioned. The number of particles in each field was assessed by fluorescence microscopy (Figure 5D).

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

Figure 5

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(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.

In our first set of experiments, we determined the duration of in vivo DANP-mediated ZNF304 silencing in an orthotopic HeyA8 mouse model. ZNF304-siRNA-DANP (300 µg/Kg body weight) was administered as a single intravenous injection 2 weeks after tumor inoculation. Groups of mice were euthanized on day 3, 7, and 14 after injection. Tumors were collected and analyzed by immunoblotting to determine ZNF304 protein expression levels. We demonstrated that ZNF304 protein silencing started at day 3 and continued up to 14 days after a single administration of ZNF304-siRNA-DANP (Figure 5E).

Next, 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), DANP-ZNF304 siRNA (n=6) or no treatment (n=2) (Figure 5F) and serum was collected after 72h using cardiac puncture. A Luminex assay designed to detect 12 pro-inflammatory cytokines (LIX, MIP-2, KC, IL-10, IL-6, IL-2, IL-1β, IL-1β, M-CSF, TNFα, GM-CSF, G-CSF) was used. The results obtained did not show any significant increase in these cytokines. Tissue samples were also obtained for post-mortem histopathology studies (brain, spleen, liver and kidney). H&E staining of the various tissues were analyzed by our Histopathology Core’s veterinary pathologist; no inflammatory changes were observed in the tissues studied (Supplementary Figure 12). Blood chemistries to assess liver (ALT and Alk Phosp) and kidney function (BUN and S. Creatinine) were also conducted (Supplementary Figure 11); hematologic profile (Complete Blood Counts, with differential and platelets: Hgb, Hct including red blood cell parameters are shown in Supplementary Table 2). All parameters studies were within normal range14. Furthermore, tissue assessment using H&E did not disclose any toxicity.

ZNF304 gene silencing leads to antitumor activity in orthotopic models of OC

On the basis of these findings, we examined the antitumor activity of weekly or biweekly ZNF304 silencing in 2 orthotopic OC mouse models, HeyA8 and SKOV3IP1. In the first model, mice were injected with HeyA8 cells to induce tumors and 1 week later were randomly assigned to 6 treatment groups (10 mice in each group): DANP alone, control siRNA-DANP, ZNF304-siRNA-DANP (150 µg/Kg body weight) administered weekly, and ZNF304-siRNA-DANP (300 µg/Kg body weight) administered biweekly, or, since paclitaxel is commonly used for OC treatment and combines effectively with many biologically targeted agents, paclitaxel only or a combination of paclitaxel plus ZNF304 siRNA-DANP (300 µg/Kg body weight, biweekly administration) (Figure 6A). Significant reductions in tumor weight were observed in the groups treated with ZNF304 siRNA-DANP weekly or biweekly. Mice treated with ZNF304 siRNA-DANP had a significantly lower tumor burden (62% reduction in tumor weight; P < 0.01) (Figure 6A, left panel) and had 50% fewer nodules than did mice treated with control siRNA-DANP (P < 0.05) (Figure 6A, right panel). Moreover, the ZNF304 siRNA-DANP treatment group had significantly fewer nodules than did the control group (P=0.0001, weekly administration; P=0.0001, biweekly administration; student’s t-test).

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

Figure 6

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(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).

In the second orthotopic model (SKOV3IP1), the treatment groups were (1) control siRNA-DANP, (2) ZNF304 siRNA-DANP, (3) control siRNA-DANP plus paclitaxel, and (4) ZNF304 siRNA-DANP plus paclitaxel (n=10/group). siRNA-DANP was administered intravenously every 2 weeks in all treatment groups. Tumors removed from mice treated with ZNF304 siRNA-DANP alone weighed 60% less than those of mice treated with DANP-control siRNA. (Figure 6B, left panel). Number of nodules was dramatically reduced in mice treated with either ZNF304 siRNA-DANP or ZNF304 siRNA-DANP plus paclitaxel (Figure 6B, right panel). The greatest reduction was observed in the group treated with both DANP-ZNF304 siRNA and paclitaxel. None of the groups in either mouse model showed decreased body weight, which indicates that the treatments were not toxic (Supplementary Figure 13). These data indicate that inhibiting ZNF304 results in antitumor activity in mouse models of OC and that the DANP delivery system is an efficient tool for in vivo gene silencing.

Given the in vitro effects of ZNF304 silencing, we performed Ki67 and CD31 staining to examine the biological effects of silencing ZNF304 on tumor cell proliferation and angiogenesis, respectively. Mice treated with ZNF304 siRNA-DANP showed significant reduction in cell proliferation compared to control group (P<0.0001) (Figure 6C). Given that ZNF304 transcriptionally regulates β1 integrin, which is required for endothelial cell adhesion, migration, and survival 15,16, we also examined the effects of ZNF304 siRNA treatment on angiogenesis. The ZNF304 siRNA-DANP treatment group had significantly reduced microvessel density compared with the control (P=0.0252)(Supplementary Figure 14). These data showed that downregulation of ZNF304 was highly associated with decreased cell proliferation and decreased microvessel density.

On the basis of efficacy data, we next examined the effects of DANP-ZNF304 siRNA treatment on tumor bearing mice survival. For these experiments, mice inoculated with OVCA-432 cells that represent HGSOC 17. Luciferase-labeled OVCA-432 cells (3×106) were injected intraperitoneally to generate tumors in nude mice. The 4 groups (n=8/group) were treated with either: 1) DANP-Control siRNA, 2) DANP-ZNF304 siRNA, 3) DANP-Control siRNA+cisplatin, and 4) DANP-ZNF304 siRNA+cisplatin. The treatment started one week after the tumor cell inoculation and the mice were monitored daily by three observers. Individual mice were euthanized on the day the core veterinarian recommended, based on moribund status. A Kaplan Meier curve was generated based on the survival duration of mice (Figure 6D). DANP-ZNF304 siRNA-based treatment significantly improved survival compared to DANP-Control siRNA treatment (n=8/group, p=0.01, Log-rank test). Our analysis showed that the median survival for DANP-Control siRNA treated group was 44 days, whereas it was not reached for the DANP-ZNF304 siRNA.

Next, we addressed whether ZNF304 silencing could directly increase the rates of anoikis in vivo. For this question, we use the OC MDAH 2774 cell line since it induces significant ascites in mice12. MDAH 2774 cells were implanted into the peritoneal cavity of nude mice, and ascites production was observed 4–6 weeks post-inoculation. Next, we analyzed the ascites for viable tumor epithelial cells using fluorescein isothiocyanate (FITC)-labeled anti-epithelial cellular adhesion molecule antibody followed by flow cytometry analysis (Figure 6E). The control group showed 16% (mean±SEM; 16.08 ± 0.7280, n=3) epithelial cell death in ascites, whereas mice treated with intravenous ZNF304 siRNA-DANP had up to 30% (mean±SEM; 30.77 ± 1.040, n=3) epithelial cell death. These results show that silencing ZNF304 significantly decreased the ability of OC cells to survive in ascites (P<0.001, Student’s t-test).

Discussion

The key finding from this study is that ZNF304 is a novel transcriptional regulator of β1 integrin. Silencing ZNF304 resulted in antitumor activity and the induction of anoikis in malignant cells both in vitro and in vivo through β1 integrin downregulation. High ZNF304 mRNA expression was associated with worse survival in OC patients. Furthermore, silencing ZNF304 enhances the anoikis rate through inhibiting inside-out integrin signaling and accordingly blocking outside-in signaling (Figure 7).

Figure 7. Schematic representation.

Figure 7

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

Integrins are crucial for normal functions of multicellular organisms and critical at each step of cancer: tumorigenesis, progression, and metastasis 18. Integrins are regulated and activated by conformational changes, clustering, and trafficking 19. These transmembrane proteins are an essential link between the extracellular matrix (ECM) and cytoplasm, and the signaling can be in 2 directions: outside-in or inside-out through the cytoplasmic β tail 19. For example, β1 integrin promotes cell survival and regulates focal adhesion, leading to tumor metastasis in many types of cancer including OC 20–23. In a recent study, moreover, Schiller and colleagues demonstrated that expression of α5β1 integrins is essential to sense the stiffness of fibronectin-based ECM, which is also critical for tumor metastasis 24,25. Furthermore, several β1 integrin-targeting strategies, such as monoclonal antibodies and peptide inhibitors, showed activity in clinical trials for cancer therapy 18,26,27. However, targeting ZNF304—the regulator of β1 integrin expression—may offer greater efficacy than targeting only activation of β1 integrin.

β1 integrin is a subunit of heterodimeric membrane adhesion receptors, and it can form heterodimers with integrin α subunits. For example, α4β1, α8β1, and αvβ1 are fibronectin-binding integrins; α3β1, α6β1, and α7β1 interact with laminin and nectin; and α1β1, α2β1, α10β1, and α11β1 bind to collagens 28,29. The first study showing the inside-out regulation of β1 integrin unraveled the control by R-Ras of the ligand-binding affinity of β1 integrin and fibronectin 30. Thus, the regulation of integrin activation and affinity was known as a transcription-independent function of the Ras-linked mitogen-activated protein kinase pathway 31,32. However, the transcriptional regulation of β1 integrin remained unknown. Here, we elucidated that the regulation of β1 integrin expression through ZNF304 is at transcriptional level.

Previous studies showed that fibronectin and β1 integrin ligation, followed by activation of cytoplasmic β subunit, promotes the invasive migration of OC cells through the ECM 33. Myosin II and FAK mediate the phosphorylation of paxillin, reinforcing the cytoskeletal ECM linkage and driving focal adhesion maturation 34. Additionally, β1 integrin-FAK signaling directs the initial proliferation of micrometastatic cancer cells disseminated in the lungs, which indicates the role of integrin-FAK signaling in the metastatic cascade 35. Thus, ZNF304 may be a regulator of this metastatic process. Correspondingly, we showed that silencing the key regulator ZNF304 decrease nodule formation, tumor growth and prolong survival in orthotopic mouse models of OC.

The functional crosstalk between cell adhesion receptors and receptor tyrosine kinases contributes to cancer cell survival 21. The interaction between ErbB1 and β1 integrin induces tumor cell detachment, migration, and metastatic potential. β1 integrin was also shown to regulate epidermal growth factor receptor signaling in lung cancer cells 36 and to mediate epidermal growth factor-induced cell invasion in OC cells 37. Furthermore, a recent study demonstrated an increased ErbB1-β1 integrin heteroassociation in high-grade astrocytomas and showed that this clinically relevant association can be targeted by molecular therapy 38. Therefore silencing ZNF304—the regulator of β1 integrin—may also inhibit epidermal growth factor receptor signaling, inhibiting cancer cell survival and slowing tumor progression.

Anoikis is a form of apoptosis in adherent nonmalignant cells caused by a lack of integrin-mediated survival signals from the ECM 39,40. However, malignant cells develop resistance to anoikis, leading to increased metastatic potential 41,42. A seminal mechanistic work on anoikis unraveled its contribution to human cancer metastasis in several different malignancies 43–45. Integrins are crucial in anoikis mechanism as major mediators of adhesion between cells and ECM proteins46,47. A recent study showed that activated integrins enhance the metastatic potential of prostate cancer cells by decreasing their sensitivity to anoikis during tumor dissemination and by increasing their interactions with ECM ligands during extravasation 48. This latter study suggested that in prostate cancer cells, β1 integrin is activated through an inside-out signaling, which also enhances its affinity for ligand binding. The interaction of β1 integrin with ECM ligands further activates β1 integrin through outside-in signaling.

RNA interference is a highly effective method for gene silencing owing to its sequence specificity; however, systemic delivery of RNA interference remains a challenge. Our group has previously developed and characterized chitosan nanoparticles for systemic delivery of siRNA 13,49. Although chitosan nanoparticles is an efficient RNA interference delivery system, weekly administration is required since the target downregulation lasts only for 7 days after a single administration. Thus, DANP prolonged silencing is a more tolerable and promotes patient compliance.

METHODS

Integrative computational analysis and patient data selection

Clinical and expression data (Level 3 Illumina HiSeqv2) for 260 patients were downloaded from The Cancer Genome Atlas portal and were used to analyze the relationship between expression of ZNF304 and overall survival as well as between expressions of ZNF304 and ITGB1. The Spearman's rank-order correlation test was applied to measure the strength of the association between ZNF304 and ITGB1 levels in patient samples in TCGA dataset.

Cell lines and culture

The immortalized non-transformed human ovarian surface epithelial cell line HIO-180 and the human epithelial OC cell lines HeyA8, MDAH 2774, SKOV3IP1, A2780PAR, and A2780CP20 were maintained as described previously50–54. Taxane resistant HeyA8MDR and SKOV3-TR cells were maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bio-Products) with or without paclitaxel (300 ng/ml for HeyA8-MDR; 150 ng/ml for SKOV3-TR). The A2780CP20 cell line was developed by sequential exposure of the A2780 cell line to increasing concentrations of cisplatin. All of the cell lines are routinely screened for Mycoplasma species (Mycoalert Mycoplasma Detection Kit, Lonza). All in vitro and in vivo experiments were conducted when cells were 70% to 80% confluent.

Western blot analysis

Western blot analysis was performed as previously reported 55,56. All antibodies used in this study and vendors are listed in Supplementary Table 4.

SiRNA constructs and delivery

SiRNAs were purchased from Qiagen or Sigma-Aldrich. A non-silencing siRNA that did not share sequence homology with any known human mRNA was used as a control for target siRNA. In vitro transient transfection was performed as described previously 55. The ZNF304 and control siRNA sequences are listed in Supplementary Table 3.

Invasion and migration assays

Cell migration and invasion assays have been described previously 57,58. For migration/invasion assays, cells were treated with either control or ZNF304 siRNA for 48 hours and incubated on migration wells for 24 hours. Migrated cells on the bottom of the wells were collected, fixed, stained, and counted by light microscopy. Cells were counted in 10 random fields (× 200 final magnification), and the average number of migrated cells was calculated; the percentage of migration was determined by setting control siRNA-treated samples as 100% migration/invasion.

RPPA

This study was conducted in The University of Texas MD Anderson Cancer Center Institution RPPA Core Facility, and the method was described previously 59.

Cell-cycle analysis

Cells were transfected with either control siRNA or ZNF304 siRNA, trypsinized and collected 72 hours post transfection. Samples were washed in phosphate-buffered saline solution (PBS) and were fixed in 75% ethanol overnight. Cells were then centrifuged and reconstituted in PBS with propidium iodide (PI; 50 µg/ml), as previously described 60. PI fluorescence was assessed by flow cytometry, and the percentage of cells in each cycle was analyzed by FlowJo software.

Chromatin immunoprecipitation assay

HeyA8 cells were cultured in 10% fetal bovine serum to ~75% confluence, and cells were cross-linked with 37% formaldehyde for 20 minutes and were incubated with glycine (0.125 M) as previously described 61. Cells were lysed, and chromatin was sonicated according to the protocol provided by the kit (EZ ChIP, Upstate Biotechnology; cat #17–371). Possible binding sites of ZNF304 in the ITGB1 promoter were predicted using an online tool (http://compbio.cs.princeton.edu/zf/). Six primer pair sets were designed using basic local alignment search tool software (National Center for Biotechnology Information). Primers used for amplification of the DNA in quantitative PCR are shown in Supplementary Table 5. Anti-ZNF304 antibody (Supplementary Table 4) was used for the chromatin immunoprecipitation assays. The Bio-Rad DNA Engine Dyad Thermal Cycler was used with the following cycling conditions: 2 minutes at 94°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 1 minute at 68°C, followed by 1 minute at 68°C.

Plasmid construction and luciferase reporter assay

Fragments containing the predicted binding sites (BS1, BS2, and BS3) were amplified from HeyA8 cell genomic DNA by PCR using primers containing SacI or HindIII restriction enzyme sites. The PCR products were purified, digested, and subsequently cloned into the same restriction site of the pGL3 control vector (Promega) downstream of the firefly luciferase reporter gene. Sequences were analyzed with a DNA BigDye Terminator sequencing kit, version 3.1 (Life Technologies) HeyA8 cells were plated in 24-well plates (60,000 cells per well) 24 hours prior to transfection with either ZNF304 siRNA or ZNF304-expressing vector (Promega). Twenty-four hours after the first transfection, cells were transfected with the luciferase reporter vectors containing BS1, BS2, or BS3 together with Renilla luciferase construct, which was used as a normalization reference. Transfections were performed with Attractene transfection reagent (Qiagen) according to the manufacturer’s instructions. Cells were lysed 48 hours after luciferase vector transfection, and activity was measured using a dual-luciferase reporter assay system (Promega) in the Veritas microplate luminometer (Turner BioSystems). Two independent experiments were performed in technical triplicates. Wild-type vectors for ZNF304 (gene ID: 57343) and ITGB1 (gene ID: 16412) were purchased from Promega (San Luis Obispo, CA). ZNF304 vector was used to generate mutant vector (7-bp deletion) by using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) using the primers in Supplementary Table 5.

In vitro anoikis

Cells were transfected with control or ZNF304 siRNA and transferred to 6-well tissue culture plates that were coated with polyhydroxyethylmethacrylate, and cells were cultured in these plates for 72 hours at 37°C in a 5% carbon dioxide atmosphere. Cells were washed with PBS and were stained with Annexin V-FITC and PI solution (50 µg/ml) containing RNase A (25 µg/ml). After incubating the pellets for 30 minutes at 37°C, we analyzed cell viability by flow cytometry.

Preparation of DANP

DANPs were prepared via ionic gelation of anionic tripolyphosphate and siRNA. Briefly, predetermined tripolyphosphate (0.25% weight/volume) and siRNA (1 µg/µl) were added to chitosan solution, and the siRNA/chitosan nanoparticles spontaneously formed under constant stirring at room temperature. After incubating the nanoparticles at 4°C for 40 minutes, we collected the siRNA/DANP by centrifugation (Thermo Biofuge) at 13,000 rpm for 40 minutes at 4°C. Chitosan nanoparticles were coated with polylactic acid polymer under probe sonication, and the organic solvent was evaporated. The pellet was washed in sterile water 3 times to isolate siRNA/DANP, which was stored at 4°C until used. For the biodistribution study, DANPs were labeled with rhodamine 6G (Sigma-Aldrich). Rhodamine 6G (0.1% weight/volume) was added to the polymer solution (chloroform) in the simple emulsion. The particles were collected and were washed 3 times to eliminate the nonencapsulated marker.

Orthotopic in vivo models of OC and tissue processing

Female athymic nude mice (NCr-nu) (8–12 weeks old) were purchased from the National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD) and were maintained as previously described 55. The MD Anderson Cancer Center Institutional Animal Care and Use Committee approved and supervised all animal studies. Mice were cared for in accordance with guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and the United States Public Health Service Policy on Human Care and Use of Laboratory Animals. To generate tumors, SKOV3IP1 cells (1 × 106), HeyA8 cells (2.5 × 105) or (3 × 106) OVCA-432 cells were injected into the peritoneal cavity, as previously described 62. For therapy experiments, 10 mice were assigned randomly to each group. This sample size was sufficient to provide 80% power for a test at significance level of 0.05. Treatments with control or ZNF304 siRNA incorporated in DANP were intravenously administered either weekly (150 µg/kg body weight) or biweekly (300 µg/kg body weight). Paclitaxel (100 µg/mouse for the HeyA8 model and 75 µg/mouse for the SKOV3 model) was injected intraperitoneally once weekly. Mice were euthanized 6 weeks after first administration in SKOV3 model and 4 weeks after first administration in HeyA8 model. After euthanasia, we recorded mouse and tumor weight, number of nodules, and distribution of tumors. Individuals who performed the necropsies were blinded to the treatment group assignments. Tissue specimens were fixed with either formalin or optimal cutting temperature medium (Miles) or were snap-frozen in liquid nitrogen. For OVCA-432 survival study, the treatment started one week after the tumor cell inoculation and the mice were monitored daily by three observers. Individual mice were euthanized on the day the core veterinarian recommended, based on moribund status.

For the biodistribution study of DANP, mice were injected peritoneally with HeyA8 cells (2.5 × 105) for tumor inoculation. When tumors were detectable, rhodamine 6G–labeled particles that contained control siRNA (150 µg/kg) were administered intravenously. After 24 hours, mice were euthanized; tumors and the major organs (brain, heart, kidney, spleen, liver, and lungs) were removed and fixed in optimal cutting temperature medium and sectioned. The organ and tumor distribution of particles was assessed by fluorescence microscopy.

In vivo anoikis

Viability of tumor cells from ascites fluid was determined by dual staining with PI and epithelial cell adhesion molecule tagged with fluorescein isothiocyanate. MDAH 2774 cells (2 × 106) were injected intraperitoneally into nude mice, and treatments started when mice developed detectable ascites. Mice were divided into 2 groups, (n=3/group) receiving a single administration of either DANP-control siRNA or DANP-ZNF304 siRNA (300 µg/kg). After 7 days, ascites fluid was drawn from the peritoneal cavity and rapidly centrifuged at 500 g for 10 minutes. Pellets were washed with a red blood cell lysis buffer and reconstituted in PBS. Suspended cells were then incubated with excited state absorption-fluorescein isothiocyanate (1:30 dilution) for 30 minutes at room temperature. After incubation, cells were washed and stained with a PI solution (50 µg/ml). Cells were then incubated for 30 minutes at 37°C and analyzed on a Gallios flow cytometer (Beckman Coulter).

Immunohistochemical analysis

Analyses of tumors cell proliferation and microvessel density were conducted by following procedures described previously13,63,64. Two investigators quantified the number of positive cells in a blinded fashion. The antibodies used and the vendors are listed in Supplementary Table 4.

Statistical analysis

Unless specified otherwise, all data are presented as the mean values ± the standard error of the mean from at least 3 independent experiments. Two-sided t tests were used to test the relationships between the means of data sets, and P values indicate the probability of the means compared, being equal with *P < 0.05, **P < 0.01 and ***P < 0.001. Student’s t tests and analysis of variance were calculated with GraphPad software. Statistical analyses were performed in R (version 3.0.1) (http:///www.r-project.org/), and P values less than 0.05 were considered statistically significant. For the analysis of RPPA results, we used the Benjamini-Hochberg multiple testing correction 65 to estimate the false discovery rate.

Supplementary Material

Suppl Data

Acknowledgments

This work was supported in part by grants from the National Institutes of Health/National Cancer Institute (R44GM086937, P50 CA093459, U54 CA151668, P50 CA083639, P50 CA098258, R21 CA180145, UH2TR000943, and U54CA96300) and from the Cancer Prevention Research Institute of Texas (RP120214).

Footnotes

SUPPLEMENTARY INFORMATION

Supplementary information includes 14 figures and 6 tables.

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