Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8

Antibodies against ZC3H8 were from AssayBiotech (#C19576) and Lifespan BioSciences (#59503) for microscopy and blotting respectively. Antibodies against PML were from Acris (#AM20293AF-N), NONO from OneWorld (#41152), COILIN monoclonal from Abnova (#B01P), COILIN polyclonal from OneWorld (#41139), CK2 from Thermo Fisher (#PA5–28686), V5 from Millipore (#AB3792), GAPDH from GeneTex (#GTX100118) or Origene (#TA802519S), and FLAG (#2368) and β-TUBULIN (#2128) were from Cell Signaling Technologies. Secondary anti-rabbit and anti-mouse HRP-linked antibodies were from Cell Signaling Technologies and fluorophore-linked antibodies were from Thermo Fisher. All antibodies were used at concentrations recommended by the commercial suppliers. 4,5,6,7-Tetrabromobenzotriazole (TBB) was purchased from Tocris. Quinalizarin was obtained from Sigma Aldrich.

Coding sequences for mouse Zc3h8 were amplified from cDNA from CV1A 03–31 mouse mammary carcinoma cells using primers For: 5’-CCCTGTCTGAGTTATGGATTTTG-3′, and Rev.: 5’-TTTACATGACTTCTTGTCAGTATC-3′. Human ZC3H8 was similarly obtained by RT-PCR of RNA from MCF7 cells using primers For: 5’-GACCTGTCTGGGTCATGGATTTG-3′ and Rev.: 5’-TTTACATGACTTCTTTTCAGTATC-3′. Amplified sequences were subsequently inserted into the TA cloning site of pcDNA 3.1 V5 His and pEF6 V5 (Thermo Fisher). Knockdown sequences shRNA A: 5’-GATCCCGGGACCGTGAACAAATTCTTCAAGAGAGAATTTGTTCACGGTCCCGTTA-3′ and its inverse complement 5’-AGCTTAACGGGACCGTGAACAAATTCTCTCTTGAAGAATTTGTTCACGGTCCCGG-3′ targeting bases 241–259 in the Zc3h8 sequence and shRNA C: 5’-GATCCCGCAAAGGGAAGCAAGTTTTTCAAGAGAAAACTTGCTCCCTTTGCGTA-3′ and the inverse complement 5’-AGCTTAACGCAAAGGGAAGCAAGTTTTCTCTTGAAAAACTTGCTTCCC TTTGCGG-3′) targeting bases 709–727 were cloned into pSilencer 4.1 (Ambion/ThermoFisher) according to the manufacturer’s directions. Negative control plasmids provided by Ambion contain sequences not found in the human, mouse, or rat genome databases. For site-directed mutagenesis of Zc3h8, the Q5 kit (New England Biolabs) was used according to manufacturer directions. Two primers were used to create T32A and T32E mutations in mouse Zc3h8: 5′- GATGAAATAGATGGTGCTGAAGTTGAAGAAACACAGACAG-3′, 5′ -GATGAAATAGATGGTGAGGAAGTTGAAGAAACACAGACAG- 3′. Each PCR reaction used the same reverse primer 5’-AATTCTTTCCTCAGGGTCGGCGGCC-3′ per the supplier’s instructions. All cloning and mutagenesis experiments were verified by DNA sequencing through Genewiz.

Mouse mammary tumor cell lines were derived from independently arising tumors in BALB/cV mice carrying the MMTV cV [20, 21]; or BALB/c mice carrying a chemically induced mammary tumor expressing a variant MMTV strain of unknown origin [22]. Tumors were excised, minced and grown in DMEM supplemented with 50% fetal bovine serum (FBS), decreased in 10% increments at each passage to a final 10% FBS. Mouse mammary cV1A 03–31, cV1A 01–51, or COMMA-D cells [23] were grown in DMEM with 10% FBS at 37 °C with 100% humidity and 5% CO₂. For treatments with TBB, cells were washed three times with DMEM without serum and incubated with either DMSO or 10 μM TBB in serum-free DMEM for 2 h in the incubator. To remove the TBB, cells were subsequently washed with DMEM full medium three times and allowed to recover for 2 h under normal conditions. Experiments using the alternative CK2 inhibitor quinalizarin were performed using the same conditions except that quinalizarin was used at 30 μM because of its reduced cell permeability. Cell transfections were done using Lipofectamine (Thermo Fisher) reagent according to manufacturer’s instructions. For stable cell line selection for RNA silencing, single colonies were selected using 500 μg/ml Geneticin treatments for 2 weeks followed by screening for expression using RT-qPCR or western blot analysis. Stable COMMA-D derivatives stably overexpressing Zc3h8 were selected using 3 μg/ml blasticidin (Thermo Fisher).

RNA was extracted from mitotically active cell cultures using Tri-Reagent (Sigma/Aldrich) according to the manufacturer’s protocol. RNA was converted to cDNA using a VILO kit (Thermo Fisher) and the product diluted in RNase-free water prior to RT-qPCR using Power-UP SYBR Green qPCR master mix (Thermo Fisher). PCR reactions were performed in triplicate, and experiments were repeated. Primers used were: Zc3h8 For: 5′- CCGCCGACCCTGAGGAAAGAATTG-3′, Rev.: 5’-GGAAGTAATGAGGGTTGAGCTGCGT-3′; Gata-3 For: 5’-AGGGACATCCTGCGCGAACTGT-3′, Rev.: 5’-CATCTTCCGGTTTCGGGTCTGG-3′; Act1 For: 5’-GACGGCCAGGTCATCACTATTG-3′, Rev.: 5’-AGGAAGGCTGGAAAAGAGCC-3′, Gapdh For: 5’-GACAACTTTGGCATTGTGG-3′, Rev.: 5’-ATGCAGGGATGATGTTCTG-3′.

Cells were grown on No. 1.5 glass coverslips for 24 h with complete medium followed by any indicated treatments. Fixatives, permeabilization solutions, and antibodies were prepared in phosphate buffered saline (1 X PBS). Coverslips were fixed for 10 min in 4% formaldehyde. Coverslips were washed in PBS three times for 5 min each. Cells on the coverslips were then permeabilized in a 0.1% Triton X-100 for 10 min. Cells were then incubated with primary antibodies for 1 h at room temperature and then washed three times in PBS for 5 min each and incubated with secondary antibodies for 1 h at room temperature. Antibody dilutions were: anti CK2 1:100 (Thermo Scientific), anti V5 1:100 (Millipore), anti ZC3H8 1:100 (Assay Biotech), anti COILIN 1:100 (monoclonal, Abnova) or 1:100 (Thermo, polyclonal), anti PML 1:2000 (Acris), anti DAXX 1:25 (Cell Signaling), anti SMN1 1:100 (Cell Signaling), and anti NARG2/ICE2 1:100 (Bioss). Cells were washed three more times in PBS for 5 min each then mounted on slides with Vectashield +DAPI and sealed with nail polish. Images were captured and processed using a Leica SP8 confocal microscope system with a Leica 63X oil immersion objective.

Wound healing assays were done using silicone inserts from Ibidi following the manufacturer’s directions. Images were captured using a 10X objective on an Olympus IX70 microscope. Statistical analysis was determined by ANOVA. Corning Biocoat Matrigel Invasion Chambers with 8.0 μm pore inserts were purchased and used according to the manufacturer’s directions. Briefly, a chemoattractant of 5% FBS in DMEM was used in the lower chamber and 2.5 × 10⁴ cells in 500 μl DMEM were added to the upper chamber and incubated for 22 h. Non-migrating cells were scraped off and migrated cells were fixed with 4% paraformaldehyde and stained with crystal violet. Cells were imaged and counted using a Leica ICC50 HD Microscope. P values were determined by Tukey post hoc. Experiments were performed in triplicate and each was repeated. Error bars represent standard deviation.

For soft agar assays, a 1 mL layer of 1.5% agarose in PBS was solidified in a 35 mm tissue culture dish and equilibrated with DMEM with 10% FBS. A subsequent layer of 2 mL of 0.5% low melting point agarose with suspended cells in DMEM and 10% FBS was layered on top and covered with 0.5 ml of liquid medium. A total of 5 × 10⁴ cells/well in each of three wells was seeded and incubated for 2 weeks. Fresh media was exchanged every 3 days. Colonies were imaged and quantified using an Olympus IX70 inverted microscope. P values were determined by Student’s t-test. For in vitro 3D spheroid growth assays, 5 × 10³ cells per well were added to eight wells per experiment and imaged every 24 h and p values were determined by ANOVA. Corning round well-bottom 96-well dishes with ultra-low attachment surface coating were used (cat. 4520). Error bars represent standard deviation. All 3-D growth assays were repeated three times.

Cell proliferation assays used the CellTiter-Glo 2.0 Luminescent Cell Viability kit (Promega). Briefly, 5 × 10³ cells (100 μL at 5 × 10⁴ cells/mL) were added to an opaque 96-well dish and incubated for 24 h. 100 μL of reagent was added to the wells and incubated for 10 min at room temperature and measured for luciferase activity using a CLARIOstar Plate Reader (BMG Labtech). Cell-free wells were used as blanks and each read included eight replicate wells per cell line per experimental condition. Measurements were taken every 24 h for 5–7 days. Each experiment was repeated three times. Statistical significance was assessed by ANOVA.

Cell lysates were extracted using RIPA Buffer (Thermo Fisher) with protease and phosphatase inhibitors (Phosphatase Inhibitor Cocktail II and Protease Inhibitor Cocktail III for mammalian cells, Research Products International). Equal amounts of protein (30 μg) were separated by SDS PAGE (10% gel) and transferred to PVDF membrane for blotting. Protein detection was done with primary and secondary antibodies and enhanced chemiluminescence reagent (Thermo Fisher). Densitometry to determine relative protein levels used ImageJ (NIH).

All experiments utilizing animals were performed according to protocols approved by Villanova University’s Animal Care and Use Committee. Subcutaneous injections of 1 × 10⁶ cells were injected into 6–8 wk. old female BALB/c mice and monitored for 8 weeks or until tumors reached 1 cm diameter. Mice were euthanized using CO₂ and tumors were dissected and weighed. Error bars represent standard deviation, and p values were determined by Student’s t- test.

Kaplan-Meier plots were generated using the Prognoscan website, Prognoscan.​org, data set GSE1456-GPL97, p value = 0.043377 [24]. The data set used is from the Breast Cancer Stockholm 1994–1996 cohort [25]. Other breast cancer data sets showed similar trends.

Supplementary Information accompanies this paper.

We identified a potential role for Zinc Finger CCCH-Type containing 8 (Zc3h8) in tumorigenesis using PCR to locate integration sites of the mouse mammary tumor virus (MMTV) in tumors of BALB/cV mice. The expected result of MMTV integration would be overexpression of the target site locus [26]. We derived a number of cell lines from mouse mammary tumors and screened these for Zc3h8 expression compared to virgin mammary gland tissue or normal mouse mammary epithelial cells. We found that 7 of 9 tested mouse mammary carcinoma cell lines have 2 fold or greater elevated expression of Zc3h8 by examining mRNA levels using RT-qPCR (Fig. 1b). One cell line expressed quite low levels of Zc3h8; the cV2A 08–32 line grows slowly in culture. Zc3h8 may therefore have a role in tumorigenesis. Online datasets also indicated a relationship between carcinogenesis, disease-free survival and ZC3H8 expression. Fig. 1c shows a sample Kaplan-Meier Plot of 159 individuals with breast cancer generated using Prognoscan [24]. High expression of ZC3H8 decreases the probability of disease-specific survival, a trend seen in many datasets for breast and other cancers. The human protein atlas (https://​www.​proteinatlas.​org/​) designates ZC3H8 as an unfavorable prognostic marker for endometrial and renal cancers [27].

Since Zc3h8 expression is elevated in many tumor cell lines, we stably transfected cells with vectors generating shRNA targeting Zc3h8 mRNA at one of two sites to generate tumor cell lines with reduced expression of Zc3h8 or negative control shRNA targets. Cell lines with very low levels of Zc3h8 expression, including CRISPR knockouts from 4 independent mouse mammary cell lines, did not survive in culture for more than a few cell divisions, however, we were able to generate cells with reduced expression that could survive. These cells have a decrease in ZC3H8 of about 25–30% as determined by RT-qPCR, western blot and densitometric analysis using ImageJ (Fig. 2a, Additional file 1: Figure S1A). Mouse mammary tumor cells (cV1A 03–31) that have knocked down expression for Zc3h8 have a slower proliferation rate than cells transfected with the control pSilencer vector as determined by cell viability assay (Fig. 2b). Strikingly, cells with reduced expression of Zc3h8 lacked the ability to form colonies in soft agar compared to control (Fig. 2c, d). Control cells transfected with an empty vector readily formed large 3D colonies suspended in semi-solid medium within 2 weeks, but no large colonies were observed in cells knocked down for Zc3h8. Only single-cells could be seen in these dishes.

Cell lines with reduced expression of Zc3h8 also have slower rates of cell migration compared to controls in wound healing assays. Mouse mammary carcinoma cells were tested for their ability to fill in a gap generated for a wound healing assay. Control cell lines migrated into the gap much more quickly than cell lines with reduced Zc3h8 (Fig. 2e). We also plated these cells in the upper chamber of a transwell invasion assay growth chamber. Control cells readily crossed the extracellular matrix gel and porous membrane to the lower chamber, however, very few cells with reduced expression of Zc3h8 were able to migrate through the membrane (Fig. 2f). Human ZC3H8 has a very similar amino acid sequence to mouse ZC3H8. Human ZC3H8 expression, however, is not affected by the shRNA designed to target the mouse mRNA. We therefore transfected cells with reduced expression of mouse Zc3h8 with a vector driving expression of human ZC3H8 and repeated the transwell invasion assay. We found that human ZC3H8 was able to rescue the knockdown phenotype and these cells showed migration efficiency similar to control cells (Fig. 2g). Knockdown of Zc3h8 expression also led to the cells’ inability to grow as spheroids, whereas the control cells with original Zc3h8 levels grew rapidly under these conditions (Fig. 2h, i ). We wanted to know if our results from studying Zc3h8 in cells in culture could translate into changes in tumor growth in vivo. To test this we injected control cells and cells with reduced expression of Zc3h8 into the mammary glands of syngeneic female BALB/c mice. Tumors were allowed to form in the mice and were removed after 8 weeks or upon reaching 1 cm in diameter. Cells with decreased expression of Zc3h8 grew more slowly in vivo, and formed significantly fewer and smaller solid tumors than control cells (Fig. 2 j, k). This indicates that while reduced expression of Zc3h8 leads to a slight decrease in cell proliferation overall, in a more challenging environment cells cannot proliferate.

Measurements of these growth properties were carried out using another cell line (cV1A 01–51) in which Zc3h8 was targeted using the same shRNA (C) and in the cV1A 03–31 cells using a different siRNA target (A). These experiments showed that cells with reduced ZC3H8 have decreased migration and invasion rates, an inability to form colonies in semi-solid medium and reduced tumor-forming ability (Additional file 1: Fig. S1 B-E). Taken with the overexpression of this gene in tumor cells, Zc3h8 appears to promote tumorigenesis. Zc3h8 may also have a role in fundamental cellular functions since we were unable to attain complete knockout of gene expression in cell culture using CRISPR.

Decreased expression of Zc3h8 in carcinoma cells decreased their growth and migration abilities and may have made these cells less aggressive. We wanted to see if non-cancerous cells could gain an aggressive phenotype if Zc3h8 was overexpressed. To test this we used COMMA-D cells, a mouse mammary epithelial cell line that has normal mammary characteristics [23]. Cells were stably transfected with mouse Zc3h8 using a strong promoter or empty vector alone and assessed for expression by RT-qPCR and western blot (Fig. 3a). COMMA-D cells with approximately four fold higher expression levels of Zc3h8 showed faster proliferation in cell viability assays (Fig. 3b). Also, cells with higher levels of ZC3H8 were capable of migrating more quickly as measured by a wound healing assay (Fig. 3c). As expected, control COMMA-D cells were not very capable of migrating through Matrigel in a transwell invasion assay, however, expression of ZC3H8 increased the ability of these cells to migrate (Fig. 3d, e). COMMA-D cells overexpressing Zc3h8 formed tumors in syngeneic BALB/c mice more rapidly than cells transfected with a control vector (Fig. 3f). The control transfected COMMA-D cells also did form some tumors, consistent with previous results [28]. Overexpression of Zc3h8 in COMMA-D cells demonstrates that these noncancerous mammary cells acquire some properties of transformed cells – faster proliferation, faster migration, invasion potential and ability to form tumors in vivo. These data complement the experiments done with tumor cells that have reduced Zc3h8 expression and the opposite phenotype.

A previous study demonstrated that ZC3H8 protein bound to an intronic regulatory region of the Gata-3 gene in T cells, decreasing Gata-3 expression and thus playing a major role in T cell development [29]. We hypothesized that ZC3H8 might have a similar role in mammary cells as Gata-3 is a major determinant of mammary cell fate [30, 31]. Ectopic overexpression of GATA-3 was shown to lead to reduced tumor outgrowth in the mammary fat pad and loss of metastatic potential of aggressive human tumor cell lines [32]. We assessed the relative levels of expression of Zc3h8 and Gata-3 in our panel of cell lines, and in knocked-down and overexpressing derivatives. Tumor cell lines demonstrated varying ratios of Zc3h8 to Gata-3 levels, while stable overexpression did not decrease Gata-3 levels (Additional file 1: Figure S1 F, G).

To elucidate the function of ZC3H8, we determined the subcellular localization of the endogenous protein using immunofluorescence confocal microscopy. In cells derived from tumors or from the normal mammary gland, ZC3H8 is located in numerous foci within the nucleoplasm (Fig. 4a and Additional file 2: Figure S2A, B). These foci resemble known nuclear domains, specifically PML bodies, Cajal Bodies, or nuclear paraspeckles. A previous study using microscopy screens to identify novel components of nuclear bodies found that ZC3H8 colocalized with p54, an essential component of nuclear paraspeckles that retain A to I –edited mRNAs [17]. However, ZC3H8 and other LEC components have been found in Cajal bodies, sites of processing and assembly of snRNAs and snRNPs [14, 15]. PML bodies contain a variety of proteins involved in genome maintenance and cellular defense responses [33].

We tested localization of ZC3H8 with PML as a marker for PML bodies, COILIN as a marker for Cajal Bodies, and NONO as a marker for paraspeckles. ZC3H8 has strong co-localization with PML and partial co-localization with COILIN (Fig. 4a, b). The localization of PML, COILIN, CK2, and ICE2 was confirmed in an additional mouse cell line, COMMA-D (Additional file 2: Figure S2A). SMN, another Cajal body marker, colocalizes with COILIN and ZC3H8 (Additional file 2: Figure S2B). ZC3H8 does not appear to co-localize with NONO (data not shown). Cajal body COILIN and additional PML body marker DAXX colocalized in HeLa cells (Additional file 2: Figure S2C), although lack of cross-species reactivity of antibodies detecting DAAX and ZC3H8 prevented us from linking all three markers in either species. Cells from lines with decreased ZC3H8 showed slightly increased numbers of PML bodies (Additional file 2: Figure S2D). These data indicate that ZC3H8 may be located in either PML bodies, Cajal Bodies or both. PML bodies participate in a surprising variety of functions, including regulation of transcription, apoptosis, DNA repair, telomere maintenance, and antiviral defense [34], while Cajal bodies are largely focused on RNA processing. As ZC3H8 contains potential nucleic acid binding motifs, its predicted structure supports these localization data.

ZC3H8 contains a consensus sequence for phosphorylation by CK2 at T32 (Fig. 1a). Interestingly, PML is also regulated by CK2 and is degraded as a result of phosphorylation via ubiquitination [35]. We decided to see if the CK2 inhibitor, TBB [36], alters ZC3H8, PML, or COILIN localization to their respective nuclear domains. TBB was most effective when cells were treated with the inhibitor in serum-free media for 2 h with 10 μM of TBB. The CK2 inhibitor had dramatic effects on the localization of both PML and ZC3H8 in treated cells. TBB treatment causes both PML and ZC3H8 to become diffuse in the nucleoplasm and associate with far fewer (albeit larger) nuclear foci (Fig. 5a). COILIN localization was unaffected (Additional file 3: Figure S3A). Additionally, ZC3H8 and PML localization can be restored by washing out TBB and allowing the cells to recover for 1 h (Fig. 5a). The number of nuclear foci containing PML or ZC3H8 were quantified for each treatment with or without TBB (Fig. 5b). An alternative CK2 inhibitor quinalizarin gave identical results (Additional file 3: Figure S3B).

We reasoned that ZC3H8 localization may be dependent either on proper PML localization or direct phosphorylation by CK2. We used site-directed mutagenesis to alter T32 within the phosphorylation site of ZC3H8. cV1A 03–31 cells were transiently transfected with wild type ZC3H8 -V5-His, T32A mutant ZC3H8 -V5-His, or T32E mutant ZC3H8 -V5-His and localized with anti-V5 immunofluorescence so that only the vector-encoded ZC3H8 product was visualized. Wild type ZC3H8 overexpression leads to the appearance of larger nuclear bodies than those in cells with only endogenous ZC3H8 expression (Fig. 5a, c). When threonine is mutated to alanine, removing the CK2 phosphorylation site, ZC3H8 is localized to PML bodies, and there is little effect on PML localization and body number (Fig. 5c, d). Interestingly, T32E ZC3H8 increases the number of PML bodies in the nucleus and they are smaller than wild type (Fig. 5c, d). Overall, PML protein levels are not changed by the ZC3H8 mutants (Additional file 3: Figure S3C). When glutamic acid with a negative charge is substituted for the wild type threonine, this protein may mimic a constitutively phosphorylated ZC3H8. This observation supports the idea that inhibition of CK2 with TBB prevents phosphorylation of ZC3H8 and alters nuclear structure and PML body number and size independent of PML phosphorylation.

ZC3H8 has also been implicated as a possible accessory protein in the little elongation complex (LEC). We co-localized one component of the complex, ICE2, also known as NARG2, with PML bodies in the nucleus (Fig. 6a). Additionally, we found that CK2 localizes to PML bodies (Fig. 6b). These data support the idea that ZC3H8 is localized to PML bodies in the nucleus by CK2 phosphorylation and works with the LEC at these transcriptionally active sites.