- Email: [email protected]
Oligonucleotide-directed STAT3 alternative splicing switch drives anti-tumorigenic outcomes in MCF10 human breast cancer cells
Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Oligonucleotide-directed STAT3 alternative splicing switch drives anti-tumorigenic outcomes in MCF10 human breast cancer cells Vincent Tano a, David A. Jans b, Marie A. Bogoyevitch a, * a b
Department of Biochemistry and Molecular Biology, Medical Building, University of Melbourne, Parkville, VIC, 3010, Australia Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Building 77, Monash University, Clayton, VIC, 3168, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2019 Accepted 7 April 2019 Available online 19 April 2019
Signal transducer and activator of transcription 3 (STAT3), a transcription factor responsive to the activation of cytokine receptors, is known for its oncogenic actions. Whilst STAT3a is the predominant spliceform in most tissues, alternative splicing of the STAT3 gene can generate a shorter STAT3b spliceform. Redirecting splicing to enhance STAT3b levels can result in tumor suppression in vivo, and so we evaluated the cellular basis underlying the anti-tumorigenic properties of STAT3b. To investigate the impact of increased STAT3b levels in cancer cells, we implemented a Morpholino-based antisense oligonucleotide strategy to modulate STAT3 spliceform expression in the MCF10CA1h cancer cells of the MCF10 series of human breast cancer cells. We employed nonsense-mediated decay (NMD) oligonucleotides and STAT3a-to-b expression switching (SWI) oligonucleotides to successfully induce STAT3 knockdown and redirect alternative splicing to increase STAT3b levels in MCF10CA1h cells, respectively. Importantly, assessment of the impacts of STAT3 splicing modulation on tumor cell biology showed that the SWI treatment significantly reduced MCF10CA1h cell growth, viability, and migration, whereas NMD treatment was without significant impact, although neither NMD nor SWI oligonucleotides significantly inhibited MCF10CA1h cell invasion through a semi-solid matrix. In conclusion, our data demonstrate that reduced breast cancer cell growth, viability and migration, but not invasion, follow the redirection of STAT3a-to-b expression switching to favour STAT3b expression. © 2019 Elsevier Inc. All rights reserved.
Keywords: STAT3 Morpholino oligonucleotide Breast cancer Proliferation Viability Invasion
1. Introduction The STAT3 transcription factor, activated through the JAK-STAT signalling pathway, is involved in many critical biological functions and complex diseases [review [1]]. Indeed, the constitutive activation of STAT3 is a cause of many human cancers, including both hematopoietic tumors such as multiple myeloma, and solid tumors like breast and lung cancers [review [1]]. Specifically, cancer-associated STAT3 functions include an enhancement of cell proliferation and survival, maintenance of pluripotency, and promotion of cancer cell migration and metastasis [reviews [2,3]]. Thus, a disruption of STAT3 signalling can inhibit tumorigenicity [4]. Conversely, STAT3 signalling is also associated with seemingly conflicting functions in initiating apoptosis, driving differentiation and growth arrest under different conditions [5]. Thus, these key roles of STAT3 reinforce its potential as a therapeutic target.
* Corresponding author. E-mail address: [email protected] (M.A. Bogoyevitch). https://doi.org/10.1016/j.bbrc.2019.04.054 0006-291X/© 2019 Elsevier Inc. All rights reserved.
The targeting of STAT3 function in cancer therapeutics has been extensively studied [review [6]]. Inhibitors targeting upstream tyrosine kinases, such as CEP-701, can block STAT3 signalling leading to tumor suppression outcomes in acute myelogenous leukemia (AML) cells [7]. However, this approach may have undesirable off-target effects because these upstream tyrosine kinases are also involved in STAT3-independent signalling pathways such as the Ras-MAPK pathway [review [1]]. Thus, therapeutic approaches directly targeting STAT3 have also been proposed [4]. In this study, we implemented a Morpholino oligonucleotidebased splicing modulation technique directly targeting the STAT3 gene transcript. Full-length STAT3a, the predominant form of STAT3 in most cell types, is an 89 kDa 770-residue protein [8]. Alternative splicing of the STAT3 gene transcript by the use of an alternative acceptor site within exon 23 of the STAT3 transcript generates a shorter 80 kDa 722-residue STAT3b splice variant with a unique C-terminal tail [9]. Importantly, redirecting STAT3a-to-b expression can induce tumor regression in human breast cancer mice xenograft models [4], although the anti-tumorigenic actions of STAT3b are not well understood. Here we delineate the impacts
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
of oligonucleotide-directed alterations in the expression of the STAT3a and STAT3b spliceforms on MCF10CA1h human breast cancer cells in vitro. The results indicate reduced breast cancer cell growth, viability and migration, but not invasion, for cells redirected to STAT3b expression, consistent with the STAT3b isoform playing an anti-tumorigenic role through impacting specific tumor cell properties. 2. Materials and methods 2.1. Cell culture, morpholino transfection and treatment MCF10A, MCF10AT, MCF10CA1h and MCF10CA1a cells were maintained in MCF10 medium (Dulbecco's modified Eagle's medium/Ham's F12 1:1 nutrient mixture (Sigma-Aldrich, USA) containing 72.5 ng/mL hydrocortisone (Sigma-Aldrich, USA), 0.01 mg/ mL bovine insulin (Sigma-Aldrich, USA), 20 ng/mL human epidermal growth factor (Life Technologies, USA), 100 ng/mL cholera toxin (Sigma-Aldrich, USA) and 1 Glutamax (Gibco, USA)) supplemented with 5% (v/v) horse serum (Life Technologies, USA) within a humidified incubator (5% CO2, 37 C). Prior to treatment, cells were seeded on tissue culture dishes and maintained in growth medium (MCF10 medium supplemented with 5% (v/v) horse serum) for at least 16 h. For Morpholino splicing modulation, medium was replaced with fresh growth medium supplemented with 16 mM Morpholino oligonucleotides (Gene Tools, USA) and 6 nM Endo-Porter transfection agent (Gene Tools, USA), and maintained for 4 days. For cytokine stimulation, cells were treated with human recombinant oncostatin M (OSM) (10 ng/mL) (Calbiochem, USA) for 0, 15 or 60 min. 2.2. Morpholino oligonucleotides for STAT3 splicing modulation Sequences for the 3 Morpholino oligonucleotides (Gene Tools, USA) used for STAT3 splicing modulation, were as per [4]. “NMD” (50 -CATTTTCTGTTCTAGATCCTGTT-30 ), a Morpholino to direct STAT3 knockdown, has an anti-sense sequence targeting the STAT3 intron-exon 6 junction; “SWI” (50 -ATTGCTGCAGGTCGTTCTGTAGG30 ), a Morpholino to direct STAT3a-to-STAT3b splicing switch, contains an anti-sense sequence targeting the intron-exon 23 junction; “INV” (50 -GGATGTCTTGCTGGACGTCGTTA-30 ), a negative control Morpholino, contains an inverse sequence of the SWI Morpholino. 2.3. Soft agar colony formation assay Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), cells were trypsinised, an aliquot stained with Trypan blue (Sigma-Aldrich, USA) to identify dead cells, and counted. Live cells (1 103 cells) were resuspended in 3 mL growth medium with 0.3% (w/v) Noble Agar (Affymetrix, USA), seeded onto a 3-mL layer of growth medium with 0.6% (w/v) agar in 6-well plates (Corning, USA), then maintained for 21 days. The sizes of cell colonies were measured using an eyepiece micrometer (Axio Imager microscope; ZEISS, Germany) and cell colonies with diameters >50 mm in the entire well were counted. 2.4. Cell lysis and immunoblot analysis Cells (8 104) were seeded in 12-well tissue culture plates (Corning, USA). Following treatment, cells were lysed in 250 mL cold RIPA buffer supplemented with Complete™ protease inhibitor mix (Roche, USA) and the total protein extract was collected after centrifugation (16,100g, 12 min, 4 C). Protein concentrations
1077
were determined by the Bio-Rad protein assay (BIO-RAD, USA). For the immunoblot analyses, proteins were resolved by SDSPAGE on 8% (w/v) polyacrylamide gels and then transferred to a PVDF membrane (Amersham Life Science, England). The following primary antibodies were used: anti-STAT3 (#610189 mouse monoclonal; BD Biosciences, USA), anti-pSTAT3 Tyr-705 (#9131 rabbit polyclonal; Cell Signalling Technology, USA), anti-pSTAT3 Ser-727 (#9134 rabbit polyclonal; Cell Signalling Technology) and anti-a-tubulin (T6074 mouse monoclonal; Sigma-Aldrich, USA). After incubation with HRP-conjugated anti-mouse or anti-rabbit antibodies (1/10,000) (Thermo Fisher Scientific, USA), immunoreactive proteins were visualised using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, USA) and the ChemiDoc™ XRSþ Imager (BIO-RAD, USA) in a signal accumulation mode. Immunoblot images were visualised in Image Lab 4.1 (BIO-RAD, USA) and densitometry analyses performed using ImageJ (National Institute of Health, USA). 2.5. Real-time quantitative polymerase chain reaction (qPCR) Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), total RNA extraction was carried out using the Purelink RNA Mini Kit (Invitrogen, USA) according to the manufacturer's protocol. cDNA was reverse transcribed from mRNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA) according to the manufacturer's protocol. qPCR was performed using 50 ng cDNA product and custom oligonucleotide primer pairs (Bioneer, Republic of Korea) with common forward (50 -GAGAAGGACATCAGCGGTAAGAC-30 ) paired with either the STAT3a transcript-specific reverse primer (50 -GGTCGTTGGTGTCACACAGATAAA-30 ) or the STAT3b transcript-specific reverse primer (50 TCAATGAATGGTGTCACACAGATAA-30 ). qPCR amplification was carried out using the QuantStudio 6 real-time PCR system and Power SYBR Green Master Mix or TaqMan Fast Advanced Master Mix (Applied Biosystems, USA) according to the manufacturers' protocols. Samples for mRNA analysis were normalised to b-actin (AssayID: ACTB, Hs99999903_m1) and GAPDH (AssayID: GAPDH, Hs99999905_m1) (Applied Biosystems, USA). The comparative threshold cycle (DDCT) method was used for data quantification and expressed as relative quantity. 2.6. Cell count and XTT assays Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). For the cell count assay, cells were trypsinised, an aliquot stained with Trypan blue (Sigma-Aldrich, USA) to identify dead cells, and counted. For the XTT assay, live cells (1 104 cells) were re-seeded in 96-well plates (Corning, USA), cultured in fresh growth medium supplemented with Morpholino for 16 h, and XTT assay performed using the Cell Proliferation Kit II (XTT) (Roche, Switzerland) according to the manufacturer's protocol. Absorbance values were measured using the iMark™ Microplate Absorbance reader (BIO-RAD, USA). Results were calculated by subtracting the A655nm values from the A490nm values. 2.7. Scratch migration assay An in vitro scratch migration assay was performed as described in Ref. [10]. Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), the cells were treated with 1 mg/mL Actinomycin D (Sigma-Aldrich, USA) (2 h) before the scratch to inhibit cell proliferation. A straight-line scratch across the cell monolayer was subsequently created with a sterile P200 pipette tip (Corning, USA)
1078
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
and the cells were then maintained in fresh growth medium supplemented with Morpholino (16 mM) and Endo-Porter (6 nM). Imaging was carried out over 24 h using a JuLI™ Stage (NanoEntek, Republic of Korea) automated cell imaging system (4 magnification, 30 min intervals). For each well, images of at least 3 distinct areas of the scratch were recorded for the full imaging period. Image analysis and scratch area quantification was carried out using ImageJ (National Institute of Health, USA). 2.8. Boyden chamber cell invasion assay The Boyden chamber cell invasion assay [11] was carried out using the BioCoat™ Matrigel Invasion Chamber (Corning, USA) according to the manufacturer's protocol. Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), medium was replaced with starvation medium (MCF10 medium supplemented with 1% (v/v) horse serum) and maintained within a humidified incubator (16 h). Following starvation, live cells (1.5 105 cells), resuspended in 0.4 mL starvation medium supplemented with Morpholino (16 mM) and Endo-Porter (6 nM), were seeded in BioCoat™ Matrigel Invasion Chamber with 8.0 mm PET Membrane (Corning, USA), and the chambers were placed in 24-well plates with 0.8 mL nutrient-rich medium (MCF10 medium supplemented with 10% (v/v) horse serum) in each well and maintained within a humidified incubator (48 h). After incubation, cells remaining on the inner surface of the chamber were removed and cells on the outer surface were fixed using 4% (w/v) paraformaldehyde at room temperature (20 min), permeabilised in 0.2% (v/v) Triton X-100/PBS at room temperature (20 min), incubated with 1% BSA/PBS (20 min) to block non-specific binding, and then stained with 250 nM DAPI/ PBS (Sigma-Aldrich, USA) at room temperature (5 min). Cells were then visualised using an Axio Imager Z1 microscope (ZEISS, Germany) and the number of cells across each entire membrane were counted. 2.9. Statistical analysis Statistical significance was calculated using Student's t-test in Microsoft Excel software (Microsoft, USA). Error bars represent standard error of the mean (SEM). 3. Results 3.1. MCF10 cancer cell tumorigenicity and cytokine-activation of STAT3 We first defined the tumorigenicity and cytokineresponsiveness of STAT3 activation in the MCF10 transformed cell line series. The MCF10 series, derived from the benign epithelial mammary gland/breast cell MCF10A [12], includes the premalignant MCF10AT [13], the malignant well-differentiated MCF10CA1h, and the malignant poorly differentiated MCF10CA1a cells, representing the progressive stages of tumor progression [14]. Using both XTT-based proliferation and soft agar colony formation assays, we observed significantly higher cell proliferation and anchorage-independent growth of the malignant MCF10CA1h and MCF10CA1a cells compared to the MCF10A and pre-malignant MCF10AT cells (Supplementary Figs. 1A and 1B), demonstrating a progression in tumorigenicity across the MCF10 series. Next, immunoblot analyses of STAT3 showed comparable levels of a single band in all four cell lines, with its size (~90 kDa) corresponding to that of STAT3a, whereas STAT3b was not detected (Supplementary Fig. 1C). In addition, pSTAT3 (Y705) levels increased following cell exposure to oncostatin M (OSM; 10 ng/mL)
in all four cell lines, demonstrating the cytokine-responsiveness of these cells (Supplementary Fig. 1C). Notably, we also detected low basal levels of pSTAT3 (S727) in the MCF10A and MCF10AT cells, but high basal levels of pSTAT3 (S727) in the malignant MCF10CA1h and MCF10CA1a cells (Supplementary Fig. 1C), consistent with previous reports [15]. Since OSM-stimulated STAT3 phosphorylation profiles were similar for the pre-malignant MCF10A and MCF10AT cells, and for the malignant MCF10CA1h and MCF10CA1a cells, we chose MCF10A cells, representing a pre-malignant state, and MCF10CA1h cells, representing a malignant state, for further study.
3.2. STAT3a/b levels are modified by morpholino-directed STAT3 splicing modulation in MCF10CA1h cells To investigate functional outcomes directed by STAT3a and STAT3b, we applied a Morpholino-directed STAT3 splicing modulation strategy [4] in the MCF10A and MCF10CA1h cells. Three Morpholino oligonucleotides were used: “NMD” to drive STAT3 knockdown through nonsense-mediated decay, “SWI” to induce a splicing switch from STAT3a-to-STAT3b, and “INV” with a nucleotide sequence the inverse of SWI to act as a negative control. In the immunoblot analysis of STAT3 expression, STAT3a was robustly detected in both lines subjected to no treatment (NT) and INV Morpholino treatment; both NMD and SWI Morpholino treatment reduced the STAT3a levels, with the SWI Morpholino treatment also resulting in the appearance of an additional STAT3b band (size z 85 kDa, lower band) (Fig. 1A). Densitometry analysis confirmed that NMD Morpholino treatment significantly decreased STAT3a levels by >80% in both MCF10A and MCF10CA1h cells (Fig. 1B). Importantly, SWI Morpholino treatment significantly increased STAT3b protein levels by ~6.5-fold and ~7.5-fold in MCF10A and MCF10CA1h cells, respectively (Fig. 1C). We also evaluated STAT3a and STAT3b transcript levels following Morpholino treatment by real-time qPCR analysis, employing custom primer pairs designed to target the common exon 20-exon 21 junction and the unique spliceform-specific exon 22-exon 23 junction (Fig. 1D). In the control cells (NT and INV), the levels of STAT3a transcripts were >4-fold higher than levels of STAT3b (Fig. 1E). NMD Morpholino treatment resulted in significant reduction of STAT3a and STAT3b transcript levels in both MCF10A and MCF10CA1h cells, when compared to the negative control INV Morpholino samples, showing up to 80% decrease in the levels of both transcripts in the MCF10CA1h cells (Fig. 1E). STAT3a-toSTAT3b splicing switch SWI Morpholino successfully induced a STAT3b transcript level increase in the MCF10CA1h (~3-fold) resulting in transcript levels close to those for STAT3a (Fig. 1E), but this was not observed in the MCF10A cells. Thus, NMD Morpholino induced STAT3 knockdown at the mRNA transcript level in both MCF10A and MCF10CA1h cells, whereas SWI Morpholino induced STAT3a-to-STAT3b expression switching (i.e. simultaneous downregulation of STAT3a and up-regulation of STAT3b) at the mRNA transcript level only in the MCF10CA1h cells. To confirm that the protein products of Morpholino-directed STAT3 splicing modulation were cytokine-responsive, we evaluated STAT3 Y705 phosphorylation by immunoblot analysis. Due to the reduced levels of STAT3 total protein in the NMD Morpholinotreated cells, levels of pSTAT3 (Y705) remain low following OSM stimulation (Fig. 2A and B). Importantly, increased pSTAT3 (Y705) levels in response to OSM stimulation were evident for STAT3b as well as STAT3a in the SWI-treated MCF10CA1h cells (Fig. 2A and B), suggesting that the expressed STAT3b protein is cytokineresponsive.
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
1079
Fig. 1. Morpholino-induced splicing modulation directs STAT3 knockdown and partial STAT3a-to-STAT3b expression switch in MCF10CA1h cells. (AeC) Immunoblot analysis of cell lysates for STAT3a and STAT3b levels in MCF10A and MCF10CA1h cells following Morpholino treatment. (D) Schematic diagram illustrating primer pairs for STAT3a and STAT3b. The complementary regions targeted by the primer pairs are indicated and the dotted lines show the exon-exon spanning regions. (E) Real-time qPCR analysis of STAT3a and STAT3b transcripts in MCF10A (left) and MCF10CA1h (right) cells following Morpholino treatment. Levels of STAT3 transcripts in each sample were expressed relative to the MCF10A NT STAT3a transcript level. (B, C, E) Results represent the mean ± SEM (n ¼ 3 independent experiments); statistically significant differences are highlighted *p 0.05, **p 0.01, ***p 0.001.
Fig. 2. Morpholino-directed partial STAT3a-to-b expression switch generates STAT3b proteins that are responsive to OSM-stimulated phosphorylation. (A) Immunoblot analyses of cell lysates for STAT3 and pSTAT3 (Y705) levels following OSM (10 ng/mL) stimulation. (B) pSTAT3a (Y705) or (C) pSTAT3b (Y705) levels were quantitated and normalised to atubulin levels. Results represent the mean ± SEM (n ¼ 3 independent experiments); statistically significant differences are highlighted *p 0.05, **p 0.01, ***p 0.001.
1080
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
3.3. Impact of morpholino-directed STAT3 splicing modulation on MCF10CA1h tumor cell biology We evaluated the tumor cell properties that may be impacted by STAT3 splicing modulation. First we assessed cell proliferation rate and cell viability. Following Morpholino treatment, live cell numbers were counted at 4 days (Fig. 3A). Live cells numbers were decreased significantly only by Morpholino-induced STAT3a-to-b switch (SWI) (Fig. 3A). We extended these observations with XTT assays as an alternative measure of cell viability. When we assayed a defined number of live cells (1 104 cells) at 16 h after reseeding, we observed lower XTT absorbance values for cells following the SWI treatment, but not NMD treatment (Fig. 3B). Taken together these results indicate that Morpholino-induced partial STAT3a-to-b expression switch, but not Morpholino-induced STAT3 knockdown, significantly decreases MCF10CA1h cell proliferation and viability. We next performed wound healing assays to assess cell migration in vitro. For cells treated with the negative control (INV) Morpholino, the scratch wound area decreased by > 80% in 20 h (Fig. 4AeC; Supplementary Video 1), with half-maximal repair (t1/ 2) of 14.0 ± 0.3 h. For NMD Morpholino-treated cells, these values were comparable (~90% recovery in 20 h with t1/2 of 12.8 ± 0.3 h). Strikingly, SWI treatment significantly reduced the MCF10CA1h migration rate (Fig. 4AeC), with a significantly larger wound size remaining compared to INV treatment at both 10 and 20 h postscratch (Fig. 4C); half-maximal recovery was also significantly longer (t1/2 of 19.2 ± 1.5 h). Thus, MCF10CA1h cell migration was significantly decreased by Morpholino-induced STAT3a-to-b expression switch. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2019.04.054 Finally, we assessed cell invasion. Following NMD or SWI Morpholino treatment, MCF10CA1h cell migration/invasion rate across a solid matrigel matrix was not significantly reduced when compared with the negative control (INV) (Fig. 4D). In addition, in soft agar colony formation assays, neither NMD nor SWI treatment significantly impacted the number of colonies formed in the agar matrix when compared with INV (Fig. 4E). Thus, MCF10CA1h cell invasion was not significantly impacted by Morpholino-directed STAT3 knockdown or STAT3a-to-b expression switch. 4. Discussion
of the STAT3 gene to favour STAT3b expression in MCF10CA1h cancer cells. Notably, our study is the first to reveal that increased STAT3b expression inhibits MCF10CA1h cell viability, proliferation and migration, whereas knockdown of STAT3a alone failed to significantly impact these cellular processes. This striking difference highlights the critical anti-tumorigenic properties of STAT3b and the need for improved understanding of the actions of the STAT3 spliceforms. Modifying alternative splicing using antisense oligonucleotides has emerged as a viable therapeutic strategy in multiple chronic diseases such as Duchenne muscular dystrophy (DMD) [16] and cancer [17]. More recently, the use of modified, stabilised Morpholino oligonucleotides has been shown to be an efficient and safe approach for directing alternative splicing modification both in vitro and in vivo [4], as seen in DMD [18]. Thus, further developments that permit stable/prolonged splicing switch of the STAT3 transcripts to favour STAT3b could provide new strategies for anti-tumor interventions. STAT3 has been described as a pleiotropic transcription factor with a diversity of direct target genes. However, gene targets of each STAT3 spliceform have not been defined. For example, while STAT3 can modulate biological functions by transcriptionally regulating a number of target genes, including A2M to drive the liver acute-phase response [19], SOCS3 to drive negative regulation of cytokine signalling [20], BCL2 to drive cell survival [21], and JUNB to drive growth arrest [22], the distinction between STAT3adirected or STAT3b-directed transcriptional regulation of these genes has not been investigated. Although the spliceforms have an identical DNA-binding domain, differences in their transactivation domains suggest that STAT3a and STAT3b drive distinct transcriptional regulatory outcomes [4,8,9]. In addition, spliceform-specific deletion studies in mice have highlighted unique physiological functions for STAT3a and STAT3b [8,23]. Thus, further attention should be directed to the possible distinct target gene regulation and downstream anti-tumorigenic outcomes when splicing is modified to enhance STAT3b expression. In conclusion, Morpholino oligonucleotide-directed splicing modification to favour STAT3b expression significantly reduces proliferation, cell viability and migration, but not invasion, in malignant MCF10CA1h cancer cells, suggesting that STAT3b can drive tumor suppression outcomes by inhibiting cell growth and motility. These results highlight the importance of STAT3b as a tumor suppressor in key cancer-associated biological processes.
Our study using the antisense oligonucleotide approach defines cellular processes that are altered upon modulating the expression
Fig. 3. Morpholino-directed partial STAT3a-to-b expression switch significantly reduces the proliferation rate of cancer MCF10CA1h cells MCF10CA1h cells were treated with either INV, NMD or SWI Morpholino (16 mM) for 4 days, followed by (A) cell count assay of the number of viable cells or (B) XTT measurements. Results represent the mean ± SEM (n ¼ 4 independent experiments); statistically significant differences are denoted *p 0.05, **p 0.01, ***p 0.001.
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
1081
Fig. 4. Morpholino-directed partial STAT3a-to-b expression switch reduces the wound healing migration rate of cancer MCF10CA1h cells. (A) Images of representative wound area following scratch of the cell monolayer in Morpholino-treated MCF10CA1h cells at the indicated times. (B) Fraction area of wound size remaining represents the fraction of unrecovered area relative to original wound size at 30-min intervals after scratch for 24 h. (C) Fraction area of wound size remaining in Morpholino-treated MCF10CA1h cells following 10 and 20 h after scratch. (D) Quantitation of the mean number of Morpholino-treated MCF10CA1h cells having invaded through the Matrigel-coated Boyden chamber after 48 h. (E) Quantitation of the mean number of colonies formed from Morpholino-treated MCF10CA1h cells in the 0.3% agarose soft agar colony formation assays after 21 days (BeE) Results represent the mean ± SEM (n ¼ 3 independent experiments); statistically significant differences are highlighted *p 0.05, ***p 0.001.
Acknowledgements The authors would like to thank Daniel Gough (Hudson Institute of Medical Research) for his assistance with the soft agar colony formation assays. This work was supported by Australian Research Council Discovery Grant DP130100804. VT is a recipient of the Melbourne International Research Scholarship at the University of Melbourne and DAJ is an NHMRC Senior Principal Research Fellow (APP1103050).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.054.
References [1] H. Yu, R. Jove, The STATs of cancer–new molecular targets come of age, Nat. Rev. Canc. 4 (2) (2004) 97e105. [2] S. Akira, Roles of STAT3 defined by tissue-specific gene targeting, Oncogene 19 (21) (2000) 2607e2611. [3] T. Hirano, K. Ishihara, M. Hibi, Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors, Oncogene 19 (21) (2000) 2548e2556. [4] F. Zammarchi, E. de Stanchina, E. Bournazou, T. Supakorndej, K. Martires, E. Riedel, A.D. Corben, J.F. Bromberg, L. Cartegni, Antitumorigenic potential of STAT3 alternative splicing modulation, Proc. Natl. Acad. Sci. U. S. A. 108 (43) (2011) 17779e17784. [5] S. Huang, Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications, Clin. Cancer Res. 13 (5) (2007) 1362e1366. [6] J. Turkson, R. Jove, STAT proteins: novel molecular targets for cancer drug discovery, Oncogene 19 (56) (2000) 6613e6626. [7] M. Levis, J. Allebach, K.F. Tse, R. Zheng, B.R. Baldwin, B.D. Smith, S. Jones-Bolin,
1082
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082 B. Ruggeri, C. Dionne, D. Small, A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo, Blood 99 (11) (2002) 3885e3891. D. Maritano, M.L. Sugrue, S. Tininini, S. Dewilde, B. Strobl, X. Fu, V. MurrayTait, R. Chiarle, V. Poli, The STAT3 isoforms alpha and beta have unique and specific functions, Nat. Immunol. 5 (4) (2004) 401e409. E. Caldenhoven, T.B. van Dijk, R. Solari, J. Armstrong, J.A. Raaijmakers, J.W. Lammers, L. Koenderman, R.P. de Groot, STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription, J. Biol. Chem. 271 (22) (1996) 13221e13227. C.C. Liang, A.Y. Park, J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro, Nat. Protoc. 2 (2) (2007) 329e333. A. Albini, Y. Iwamoto, H.K. Kleinman, G.R. Martin, S.A. Aaronson, J.M. Kozlowski, R.N. McEwan, A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res. 47 (12) (1987) 3239e3245. H.D. Soule, T.M. Maloney, S.R. Wolman, W.D. Peterson Jr., R. Brenz, C.M. McGrath, J. Russo, R.J. Pauley, R.F. Jones, S.C. Brooks, Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10, Cancer Res. 50 (18) (1990) 6075e6086. P.J. Dawson, S.R. Wolman, L. Tait, G.H. Heppner, F.R. Miller, MCF10AT: a model for the evolution of cancer from proliferative breast disease, Am. J. Pathol. 148 (1) (1996) 313e319. S.J. Santner, P.J. Dawson, L. Tait, H.D. Soule, J. Eliason, A.N. Mohamed, S.R. Wolman, G.H. Heppner, F.R. Miller, Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells, Breast Canc. Res. Treat. 65 (2) (2001) 101e110. S.H. Kim, K. Zukowski, R.F. Novak, Rapamycin effects on mTOR signaling in benign, premalignant and malignant human breast epithelial cells, Anticancer
Res. 29 (4) (2009) 1143e1150. [16] J.C. van Deutekom, A.A. Janson, I.B. Ginjaar, W.S. Frankhuizen, A. Aartsma-Rus, M. Bremmer-Bout, J.T. den Dunnen, K. Koop, A.J. van der Kooi, N.M. Goemans, et al., Local dystrophin restoration with antisense oligonucleotide PRO051, N. Engl. J. Med. 357 (26) (2007) 2677e2686. [17] T. Williams, R. Kole, Analysis of prostate-specific membrane antigen splice variants in LNCap cells, Oligonucleotides 16 (2) (2006) 186e195. [18] M. Kinali, V. Arechavala-Gomeza, L. Feng, S. Cirak, D. Hunt, C. Adkin, M. Guglieri, E. Ashton, S. Abbs, P. Nihoyannopoulos, et al., Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study, Lancet Neurol. 8 (10) (2009) 918e928. [19] U.M. Wegenka, J. Buschmann, C. Lutticken, P.C. Heinrich, F. Horn, Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level, Mol. Cell Biol. 13 (1) (1993) 276e288. [20] L. Zhang, D.B. Badgwell, J.J. Bevers 3rd, K. Schlessinger, P.J. Murray, D.E. Levy, S.S. Watowich, IL-6 signaling via the STAT3/SOCS3 pathway: functional analysis of the conserved STAT3 N-domain, Mol. Cell. Biochem. 288 (1e2) (2006) 179e189. [21] S. Bhattacharya, R.M. Ray, L.R. Johnson, STAT3-mediated transcription of Bcl-2, Mcl-1 and c-IAP2 prevents apoptosis in polyamine-depleted cells, Biochem. J. 392 (Pt 2) (2005) 335e344. [22] Y. Yamanaka, K. Nakajima, T. Fukada, M. Hibi, T. Hirano, Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation, EMBO J. 15 (7) (1996) 1557e1565. [23] S. Dewilde, A. Vercelli, R. Chiarle, V. Poli, Of alphas and betas: distinct and overlapping functions of STAT3 isoforms, Front. Biosci. 13 (2008) 6501e6514.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Oligonucleotide-directed STAT3 alternative splicing switch drives anti-tumorigenic outcomes in MCF10 human breast cancer cells Vincent Tano a, David A. Jans b, Marie A. Bogoyevitch a, * a b
Department of Biochemistry and Molecular Biology, Medical Building, University of Melbourne, Parkville, VIC, 3010, Australia Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Building 77, Monash University, Clayton, VIC, 3168, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2019 Accepted 7 April 2019 Available online 19 April 2019
Signal transducer and activator of transcription 3 (STAT3), a transcription factor responsive to the activation of cytokine receptors, is known for its oncogenic actions. Whilst STAT3a is the predominant spliceform in most tissues, alternative splicing of the STAT3 gene can generate a shorter STAT3b spliceform. Redirecting splicing to enhance STAT3b levels can result in tumor suppression in vivo, and so we evaluated the cellular basis underlying the anti-tumorigenic properties of STAT3b. To investigate the impact of increased STAT3b levels in cancer cells, we implemented a Morpholino-based antisense oligonucleotide strategy to modulate STAT3 spliceform expression in the MCF10CA1h cancer cells of the MCF10 series of human breast cancer cells. We employed nonsense-mediated decay (NMD) oligonucleotides and STAT3a-to-b expression switching (SWI) oligonucleotides to successfully induce STAT3 knockdown and redirect alternative splicing to increase STAT3b levels in MCF10CA1h cells, respectively. Importantly, assessment of the impacts of STAT3 splicing modulation on tumor cell biology showed that the SWI treatment significantly reduced MCF10CA1h cell growth, viability, and migration, whereas NMD treatment was without significant impact, although neither NMD nor SWI oligonucleotides significantly inhibited MCF10CA1h cell invasion through a semi-solid matrix. In conclusion, our data demonstrate that reduced breast cancer cell growth, viability and migration, but not invasion, follow the redirection of STAT3a-to-b expression switching to favour STAT3b expression. © 2019 Elsevier Inc. All rights reserved.
Keywords: STAT3 Morpholino oligonucleotide Breast cancer Proliferation Viability Invasion
1. Introduction The STAT3 transcription factor, activated through the JAK-STAT signalling pathway, is involved in many critical biological functions and complex diseases [review [1]]. Indeed, the constitutive activation of STAT3 is a cause of many human cancers, including both hematopoietic tumors such as multiple myeloma, and solid tumors like breast and lung cancers [review [1]]. Specifically, cancer-associated STAT3 functions include an enhancement of cell proliferation and survival, maintenance of pluripotency, and promotion of cancer cell migration and metastasis [reviews [2,3]]. Thus, a disruption of STAT3 signalling can inhibit tumorigenicity [4]. Conversely, STAT3 signalling is also associated with seemingly conflicting functions in initiating apoptosis, driving differentiation and growth arrest under different conditions [5]. Thus, these key roles of STAT3 reinforce its potential as a therapeutic target.
* Corresponding author. E-mail address: [email protected] (M.A. Bogoyevitch). https://doi.org/10.1016/j.bbrc.2019.04.054 0006-291X/© 2019 Elsevier Inc. All rights reserved.
The targeting of STAT3 function in cancer therapeutics has been extensively studied [review [6]]. Inhibitors targeting upstream tyrosine kinases, such as CEP-701, can block STAT3 signalling leading to tumor suppression outcomes in acute myelogenous leukemia (AML) cells [7]. However, this approach may have undesirable off-target effects because these upstream tyrosine kinases are also involved in STAT3-independent signalling pathways such as the Ras-MAPK pathway [review [1]]. Thus, therapeutic approaches directly targeting STAT3 have also been proposed [4]. In this study, we implemented a Morpholino oligonucleotidebased splicing modulation technique directly targeting the STAT3 gene transcript. Full-length STAT3a, the predominant form of STAT3 in most cell types, is an 89 kDa 770-residue protein [8]. Alternative splicing of the STAT3 gene transcript by the use of an alternative acceptor site within exon 23 of the STAT3 transcript generates a shorter 80 kDa 722-residue STAT3b splice variant with a unique C-terminal tail [9]. Importantly, redirecting STAT3a-to-b expression can induce tumor regression in human breast cancer mice xenograft models [4], although the anti-tumorigenic actions of STAT3b are not well understood. Here we delineate the impacts
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
of oligonucleotide-directed alterations in the expression of the STAT3a and STAT3b spliceforms on MCF10CA1h human breast cancer cells in vitro. The results indicate reduced breast cancer cell growth, viability and migration, but not invasion, for cells redirected to STAT3b expression, consistent with the STAT3b isoform playing an anti-tumorigenic role through impacting specific tumor cell properties. 2. Materials and methods 2.1. Cell culture, morpholino transfection and treatment MCF10A, MCF10AT, MCF10CA1h and MCF10CA1a cells were maintained in MCF10 medium (Dulbecco's modified Eagle's medium/Ham's F12 1:1 nutrient mixture (Sigma-Aldrich, USA) containing 72.5 ng/mL hydrocortisone (Sigma-Aldrich, USA), 0.01 mg/ mL bovine insulin (Sigma-Aldrich, USA), 20 ng/mL human epidermal growth factor (Life Technologies, USA), 100 ng/mL cholera toxin (Sigma-Aldrich, USA) and 1 Glutamax (Gibco, USA)) supplemented with 5% (v/v) horse serum (Life Technologies, USA) within a humidified incubator (5% CO2, 37 C). Prior to treatment, cells were seeded on tissue culture dishes and maintained in growth medium (MCF10 medium supplemented with 5% (v/v) horse serum) for at least 16 h. For Morpholino splicing modulation, medium was replaced with fresh growth medium supplemented with 16 mM Morpholino oligonucleotides (Gene Tools, USA) and 6 nM Endo-Porter transfection agent (Gene Tools, USA), and maintained for 4 days. For cytokine stimulation, cells were treated with human recombinant oncostatin M (OSM) (10 ng/mL) (Calbiochem, USA) for 0, 15 or 60 min. 2.2. Morpholino oligonucleotides for STAT3 splicing modulation Sequences for the 3 Morpholino oligonucleotides (Gene Tools, USA) used for STAT3 splicing modulation, were as per [4]. “NMD” (50 -CATTTTCTGTTCTAGATCCTGTT-30 ), a Morpholino to direct STAT3 knockdown, has an anti-sense sequence targeting the STAT3 intron-exon 6 junction; “SWI” (50 -ATTGCTGCAGGTCGTTCTGTAGG30 ), a Morpholino to direct STAT3a-to-STAT3b splicing switch, contains an anti-sense sequence targeting the intron-exon 23 junction; “INV” (50 -GGATGTCTTGCTGGACGTCGTTA-30 ), a negative control Morpholino, contains an inverse sequence of the SWI Morpholino. 2.3. Soft agar colony formation assay Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), cells were trypsinised, an aliquot stained with Trypan blue (Sigma-Aldrich, USA) to identify dead cells, and counted. Live cells (1 103 cells) were resuspended in 3 mL growth medium with 0.3% (w/v) Noble Agar (Affymetrix, USA), seeded onto a 3-mL layer of growth medium with 0.6% (w/v) agar in 6-well plates (Corning, USA), then maintained for 21 days. The sizes of cell colonies were measured using an eyepiece micrometer (Axio Imager microscope; ZEISS, Germany) and cell colonies with diameters >50 mm in the entire well were counted. 2.4. Cell lysis and immunoblot analysis Cells (8 104) were seeded in 12-well tissue culture plates (Corning, USA). Following treatment, cells were lysed in 250 mL cold RIPA buffer supplemented with Complete™ protease inhibitor mix (Roche, USA) and the total protein extract was collected after centrifugation (16,100g, 12 min, 4 C). Protein concentrations
1077
were determined by the Bio-Rad protein assay (BIO-RAD, USA). For the immunoblot analyses, proteins were resolved by SDSPAGE on 8% (w/v) polyacrylamide gels and then transferred to a PVDF membrane (Amersham Life Science, England). The following primary antibodies were used: anti-STAT3 (#610189 mouse monoclonal; BD Biosciences, USA), anti-pSTAT3 Tyr-705 (#9131 rabbit polyclonal; Cell Signalling Technology, USA), anti-pSTAT3 Ser-727 (#9134 rabbit polyclonal; Cell Signalling Technology) and anti-a-tubulin (T6074 mouse monoclonal; Sigma-Aldrich, USA). After incubation with HRP-conjugated anti-mouse or anti-rabbit antibodies (1/10,000) (Thermo Fisher Scientific, USA), immunoreactive proteins were visualised using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, USA) and the ChemiDoc™ XRSþ Imager (BIO-RAD, USA) in a signal accumulation mode. Immunoblot images were visualised in Image Lab 4.1 (BIO-RAD, USA) and densitometry analyses performed using ImageJ (National Institute of Health, USA). 2.5. Real-time quantitative polymerase chain reaction (qPCR) Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), total RNA extraction was carried out using the Purelink RNA Mini Kit (Invitrogen, USA) according to the manufacturer's protocol. cDNA was reverse transcribed from mRNA using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, USA) according to the manufacturer's protocol. qPCR was performed using 50 ng cDNA product and custom oligonucleotide primer pairs (Bioneer, Republic of Korea) with common forward (50 -GAGAAGGACATCAGCGGTAAGAC-30 ) paired with either the STAT3a transcript-specific reverse primer (50 -GGTCGTTGGTGTCACACAGATAAA-30 ) or the STAT3b transcript-specific reverse primer (50 TCAATGAATGGTGTCACACAGATAA-30 ). qPCR amplification was carried out using the QuantStudio 6 real-time PCR system and Power SYBR Green Master Mix or TaqMan Fast Advanced Master Mix (Applied Biosystems, USA) according to the manufacturers' protocols. Samples for mRNA analysis were normalised to b-actin (AssayID: ACTB, Hs99999903_m1) and GAPDH (AssayID: GAPDH, Hs99999905_m1) (Applied Biosystems, USA). The comparative threshold cycle (DDCT) method was used for data quantification and expressed as relative quantity. 2.6. Cell count and XTT assays Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). For the cell count assay, cells were trypsinised, an aliquot stained with Trypan blue (Sigma-Aldrich, USA) to identify dead cells, and counted. For the XTT assay, live cells (1 104 cells) were re-seeded in 96-well plates (Corning, USA), cultured in fresh growth medium supplemented with Morpholino for 16 h, and XTT assay performed using the Cell Proliferation Kit II (XTT) (Roche, Switzerland) according to the manufacturer's protocol. Absorbance values were measured using the iMark™ Microplate Absorbance reader (BIO-RAD, USA). Results were calculated by subtracting the A655nm values from the A490nm values. 2.7. Scratch migration assay An in vitro scratch migration assay was performed as described in Ref. [10]. Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), the cells were treated with 1 mg/mL Actinomycin D (Sigma-Aldrich, USA) (2 h) before the scratch to inhibit cell proliferation. A straight-line scratch across the cell monolayer was subsequently created with a sterile P200 pipette tip (Corning, USA)
1078
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
and the cells were then maintained in fresh growth medium supplemented with Morpholino (16 mM) and Endo-Porter (6 nM). Imaging was carried out over 24 h using a JuLI™ Stage (NanoEntek, Republic of Korea) automated cell imaging system (4 magnification, 30 min intervals). For each well, images of at least 3 distinct areas of the scratch were recorded for the full imaging period. Image analysis and scratch area quantification was carried out using ImageJ (National Institute of Health, USA). 2.8. Boyden chamber cell invasion assay The Boyden chamber cell invasion assay [11] was carried out using the BioCoat™ Matrigel Invasion Chamber (Corning, USA) according to the manufacturer's protocol. Cells (8 104 cells) were seeded in 12-well tissue culture plates (Corning, USA). Following Morpholino oligonucleotide treatment (4 days), medium was replaced with starvation medium (MCF10 medium supplemented with 1% (v/v) horse serum) and maintained within a humidified incubator (16 h). Following starvation, live cells (1.5 105 cells), resuspended in 0.4 mL starvation medium supplemented with Morpholino (16 mM) and Endo-Porter (6 nM), were seeded in BioCoat™ Matrigel Invasion Chamber with 8.0 mm PET Membrane (Corning, USA), and the chambers were placed in 24-well plates with 0.8 mL nutrient-rich medium (MCF10 medium supplemented with 10% (v/v) horse serum) in each well and maintained within a humidified incubator (48 h). After incubation, cells remaining on the inner surface of the chamber were removed and cells on the outer surface were fixed using 4% (w/v) paraformaldehyde at room temperature (20 min), permeabilised in 0.2% (v/v) Triton X-100/PBS at room temperature (20 min), incubated with 1% BSA/PBS (20 min) to block non-specific binding, and then stained with 250 nM DAPI/ PBS (Sigma-Aldrich, USA) at room temperature (5 min). Cells were then visualised using an Axio Imager Z1 microscope (ZEISS, Germany) and the number of cells across each entire membrane were counted. 2.9. Statistical analysis Statistical significance was calculated using Student's t-test in Microsoft Excel software (Microsoft, USA). Error bars represent standard error of the mean (SEM). 3. Results 3.1. MCF10 cancer cell tumorigenicity and cytokine-activation of STAT3 We first defined the tumorigenicity and cytokineresponsiveness of STAT3 activation in the MCF10 transformed cell line series. The MCF10 series, derived from the benign epithelial mammary gland/breast cell MCF10A [12], includes the premalignant MCF10AT [13], the malignant well-differentiated MCF10CA1h, and the malignant poorly differentiated MCF10CA1a cells, representing the progressive stages of tumor progression [14]. Using both XTT-based proliferation and soft agar colony formation assays, we observed significantly higher cell proliferation and anchorage-independent growth of the malignant MCF10CA1h and MCF10CA1a cells compared to the MCF10A and pre-malignant MCF10AT cells (Supplementary Figs. 1A and 1B), demonstrating a progression in tumorigenicity across the MCF10 series. Next, immunoblot analyses of STAT3 showed comparable levels of a single band in all four cell lines, with its size (~90 kDa) corresponding to that of STAT3a, whereas STAT3b was not detected (Supplementary Fig. 1C). In addition, pSTAT3 (Y705) levels increased following cell exposure to oncostatin M (OSM; 10 ng/mL)
in all four cell lines, demonstrating the cytokine-responsiveness of these cells (Supplementary Fig. 1C). Notably, we also detected low basal levels of pSTAT3 (S727) in the MCF10A and MCF10AT cells, but high basal levels of pSTAT3 (S727) in the malignant MCF10CA1h and MCF10CA1a cells (Supplementary Fig. 1C), consistent with previous reports [15]. Since OSM-stimulated STAT3 phosphorylation profiles were similar for the pre-malignant MCF10A and MCF10AT cells, and for the malignant MCF10CA1h and MCF10CA1a cells, we chose MCF10A cells, representing a pre-malignant state, and MCF10CA1h cells, representing a malignant state, for further study.
3.2. STAT3a/b levels are modified by morpholino-directed STAT3 splicing modulation in MCF10CA1h cells To investigate functional outcomes directed by STAT3a and STAT3b, we applied a Morpholino-directed STAT3 splicing modulation strategy [4] in the MCF10A and MCF10CA1h cells. Three Morpholino oligonucleotides were used: “NMD” to drive STAT3 knockdown through nonsense-mediated decay, “SWI” to induce a splicing switch from STAT3a-to-STAT3b, and “INV” with a nucleotide sequence the inverse of SWI to act as a negative control. In the immunoblot analysis of STAT3 expression, STAT3a was robustly detected in both lines subjected to no treatment (NT) and INV Morpholino treatment; both NMD and SWI Morpholino treatment reduced the STAT3a levels, with the SWI Morpholino treatment also resulting in the appearance of an additional STAT3b band (size z 85 kDa, lower band) (Fig. 1A). Densitometry analysis confirmed that NMD Morpholino treatment significantly decreased STAT3a levels by >80% in both MCF10A and MCF10CA1h cells (Fig. 1B). Importantly, SWI Morpholino treatment significantly increased STAT3b protein levels by ~6.5-fold and ~7.5-fold in MCF10A and MCF10CA1h cells, respectively (Fig. 1C). We also evaluated STAT3a and STAT3b transcript levels following Morpholino treatment by real-time qPCR analysis, employing custom primer pairs designed to target the common exon 20-exon 21 junction and the unique spliceform-specific exon 22-exon 23 junction (Fig. 1D). In the control cells (NT and INV), the levels of STAT3a transcripts were >4-fold higher than levels of STAT3b (Fig. 1E). NMD Morpholino treatment resulted in significant reduction of STAT3a and STAT3b transcript levels in both MCF10A and MCF10CA1h cells, when compared to the negative control INV Morpholino samples, showing up to 80% decrease in the levels of both transcripts in the MCF10CA1h cells (Fig. 1E). STAT3a-toSTAT3b splicing switch SWI Morpholino successfully induced a STAT3b transcript level increase in the MCF10CA1h (~3-fold) resulting in transcript levels close to those for STAT3a (Fig. 1E), but this was not observed in the MCF10A cells. Thus, NMD Morpholino induced STAT3 knockdown at the mRNA transcript level in both MCF10A and MCF10CA1h cells, whereas SWI Morpholino induced STAT3a-to-STAT3b expression switching (i.e. simultaneous downregulation of STAT3a and up-regulation of STAT3b) at the mRNA transcript level only in the MCF10CA1h cells. To confirm that the protein products of Morpholino-directed STAT3 splicing modulation were cytokine-responsive, we evaluated STAT3 Y705 phosphorylation by immunoblot analysis. Due to the reduced levels of STAT3 total protein in the NMD Morpholinotreated cells, levels of pSTAT3 (Y705) remain low following OSM stimulation (Fig. 2A and B). Importantly, increased pSTAT3 (Y705) levels in response to OSM stimulation were evident for STAT3b as well as STAT3a in the SWI-treated MCF10CA1h cells (Fig. 2A and B), suggesting that the expressed STAT3b protein is cytokineresponsive.
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
1079
Fig. 1. Morpholino-induced splicing modulation directs STAT3 knockdown and partial STAT3a-to-STAT3b expression switch in MCF10CA1h cells. (AeC) Immunoblot analysis of cell lysates for STAT3a and STAT3b levels in MCF10A and MCF10CA1h cells following Morpholino treatment. (D) Schematic diagram illustrating primer pairs for STAT3a and STAT3b. The complementary regions targeted by the primer pairs are indicated and the dotted lines show the exon-exon spanning regions. (E) Real-time qPCR analysis of STAT3a and STAT3b transcripts in MCF10A (left) and MCF10CA1h (right) cells following Morpholino treatment. Levels of STAT3 transcripts in each sample were expressed relative to the MCF10A NT STAT3a transcript level. (B, C, E) Results represent the mean ± SEM (n ¼ 3 independent experiments); statistically significant differences are highlighted *p 0.05, **p 0.01, ***p 0.001.
Fig. 2. Morpholino-directed partial STAT3a-to-b expression switch generates STAT3b proteins that are responsive to OSM-stimulated phosphorylation. (A) Immunoblot analyses of cell lysates for STAT3 and pSTAT3 (Y705) levels following OSM (10 ng/mL) stimulation. (B) pSTAT3a (Y705) or (C) pSTAT3b (Y705) levels were quantitated and normalised to atubulin levels. Results represent the mean ± SEM (n ¼ 3 independent experiments); statistically significant differences are highlighted *p 0.05, **p 0.01, ***p 0.001.
1080
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
3.3. Impact of morpholino-directed STAT3 splicing modulation on MCF10CA1h tumor cell biology We evaluated the tumor cell properties that may be impacted by STAT3 splicing modulation. First we assessed cell proliferation rate and cell viability. Following Morpholino treatment, live cell numbers were counted at 4 days (Fig. 3A). Live cells numbers were decreased significantly only by Morpholino-induced STAT3a-to-b switch (SWI) (Fig. 3A). We extended these observations with XTT assays as an alternative measure of cell viability. When we assayed a defined number of live cells (1 104 cells) at 16 h after reseeding, we observed lower XTT absorbance values for cells following the SWI treatment, but not NMD treatment (Fig. 3B). Taken together these results indicate that Morpholino-induced partial STAT3a-to-b expression switch, but not Morpholino-induced STAT3 knockdown, significantly decreases MCF10CA1h cell proliferation and viability. We next performed wound healing assays to assess cell migration in vitro. For cells treated with the negative control (INV) Morpholino, the scratch wound area decreased by > 80% in 20 h (Fig. 4AeC; Supplementary Video 1), with half-maximal repair (t1/ 2) of 14.0 ± 0.3 h. For NMD Morpholino-treated cells, these values were comparable (~90% recovery in 20 h with t1/2 of 12.8 ± 0.3 h). Strikingly, SWI treatment significantly reduced the MCF10CA1h migration rate (Fig. 4AeC), with a significantly larger wound size remaining compared to INV treatment at both 10 and 20 h postscratch (Fig. 4C); half-maximal recovery was also significantly longer (t1/2 of 19.2 ± 1.5 h). Thus, MCF10CA1h cell migration was significantly decreased by Morpholino-induced STAT3a-to-b expression switch. Supplementary video related to this article can be found at https://doi.org/10.1016/j.bbrc.2019.04.054 Finally, we assessed cell invasion. Following NMD or SWI Morpholino treatment, MCF10CA1h cell migration/invasion rate across a solid matrigel matrix was not significantly reduced when compared with the negative control (INV) (Fig. 4D). In addition, in soft agar colony formation assays, neither NMD nor SWI treatment significantly impacted the number of colonies formed in the agar matrix when compared with INV (Fig. 4E). Thus, MCF10CA1h cell invasion was not significantly impacted by Morpholino-directed STAT3 knockdown or STAT3a-to-b expression switch. 4. Discussion
of the STAT3 gene to favour STAT3b expression in MCF10CA1h cancer cells. Notably, our study is the first to reveal that increased STAT3b expression inhibits MCF10CA1h cell viability, proliferation and migration, whereas knockdown of STAT3a alone failed to significantly impact these cellular processes. This striking difference highlights the critical anti-tumorigenic properties of STAT3b and the need for improved understanding of the actions of the STAT3 spliceforms. Modifying alternative splicing using antisense oligonucleotides has emerged as a viable therapeutic strategy in multiple chronic diseases such as Duchenne muscular dystrophy (DMD) [16] and cancer [17]. More recently, the use of modified, stabilised Morpholino oligonucleotides has been shown to be an efficient and safe approach for directing alternative splicing modification both in vitro and in vivo [4], as seen in DMD [18]. Thus, further developments that permit stable/prolonged splicing switch of the STAT3 transcripts to favour STAT3b could provide new strategies for anti-tumor interventions. STAT3 has been described as a pleiotropic transcription factor with a diversity of direct target genes. However, gene targets of each STAT3 spliceform have not been defined. For example, while STAT3 can modulate biological functions by transcriptionally regulating a number of target genes, including A2M to drive the liver acute-phase response [19], SOCS3 to drive negative regulation of cytokine signalling [20], BCL2 to drive cell survival [21], and JUNB to drive growth arrest [22], the distinction between STAT3adirected or STAT3b-directed transcriptional regulation of these genes has not been investigated. Although the spliceforms have an identical DNA-binding domain, differences in their transactivation domains suggest that STAT3a and STAT3b drive distinct transcriptional regulatory outcomes [4,8,9]. In addition, spliceform-specific deletion studies in mice have highlighted unique physiological functions for STAT3a and STAT3b [8,23]. Thus, further attention should be directed to the possible distinct target gene regulation and downstream anti-tumorigenic outcomes when splicing is modified to enhance STAT3b expression. In conclusion, Morpholino oligonucleotide-directed splicing modification to favour STAT3b expression significantly reduces proliferation, cell viability and migration, but not invasion, in malignant MCF10CA1h cancer cells, suggesting that STAT3b can drive tumor suppression outcomes by inhibiting cell growth and motility. These results highlight the importance of STAT3b as a tumor suppressor in key cancer-associated biological processes.
Our study using the antisense oligonucleotide approach defines cellular processes that are altered upon modulating the expression
Fig. 3. Morpholino-directed partial STAT3a-to-b expression switch significantly reduces the proliferation rate of cancer MCF10CA1h cells MCF10CA1h cells were treated with either INV, NMD or SWI Morpholino (16 mM) for 4 days, followed by (A) cell count assay of the number of viable cells or (B) XTT measurements. Results represent the mean ± SEM (n ¼ 4 independent experiments); statistically significant differences are denoted *p 0.05, **p 0.01, ***p 0.001.
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082
1081
Fig. 4. Morpholino-directed partial STAT3a-to-b expression switch reduces the wound healing migration rate of cancer MCF10CA1h cells. (A) Images of representative wound area following scratch of the cell monolayer in Morpholino-treated MCF10CA1h cells at the indicated times. (B) Fraction area of wound size remaining represents the fraction of unrecovered area relative to original wound size at 30-min intervals after scratch for 24 h. (C) Fraction area of wound size remaining in Morpholino-treated MCF10CA1h cells following 10 and 20 h after scratch. (D) Quantitation of the mean number of Morpholino-treated MCF10CA1h cells having invaded through the Matrigel-coated Boyden chamber after 48 h. (E) Quantitation of the mean number of colonies formed from Morpholino-treated MCF10CA1h cells in the 0.3% agarose soft agar colony formation assays after 21 days (BeE) Results represent the mean ± SEM (n ¼ 3 independent experiments); statistically significant differences are highlighted *p 0.05, ***p 0.001.
Acknowledgements The authors would like to thank Daniel Gough (Hudson Institute of Medical Research) for his assistance with the soft agar colony formation assays. This work was supported by Australian Research Council Discovery Grant DP130100804. VT is a recipient of the Melbourne International Research Scholarship at the University of Melbourne and DAJ is an NHMRC Senior Principal Research Fellow (APP1103050).
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.054.
References [1] H. Yu, R. Jove, The STATs of cancer–new molecular targets come of age, Nat. Rev. Canc. 4 (2) (2004) 97e105. [2] S. Akira, Roles of STAT3 defined by tissue-specific gene targeting, Oncogene 19 (21) (2000) 2607e2611. [3] T. Hirano, K. Ishihara, M. Hibi, Roles of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptors, Oncogene 19 (21) (2000) 2548e2556. [4] F. Zammarchi, E. de Stanchina, E. Bournazou, T. Supakorndej, K. Martires, E. Riedel, A.D. Corben, J.F. Bromberg, L. Cartegni, Antitumorigenic potential of STAT3 alternative splicing modulation, Proc. Natl. Acad. Sci. U. S. A. 108 (43) (2011) 17779e17784. [5] S. Huang, Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications, Clin. Cancer Res. 13 (5) (2007) 1362e1366. [6] J. Turkson, R. Jove, STAT proteins: novel molecular targets for cancer drug discovery, Oncogene 19 (56) (2000) 6613e6626. [7] M. Levis, J. Allebach, K.F. Tse, R. Zheng, B.R. Baldwin, B.D. Smith, S. Jones-Bolin,
1082
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
V. Tano et al. / Biochemical and Biophysical Research Communications 513 (2019) 1076e1082 B. Ruggeri, C. Dionne, D. Small, A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo, Blood 99 (11) (2002) 3885e3891. D. Maritano, M.L. Sugrue, S. Tininini, S. Dewilde, B. Strobl, X. Fu, V. MurrayTait, R. Chiarle, V. Poli, The STAT3 isoforms alpha and beta have unique and specific functions, Nat. Immunol. 5 (4) (2004) 401e409. E. Caldenhoven, T.B. van Dijk, R. Solari, J. Armstrong, J.A. Raaijmakers, J.W. Lammers, L. Koenderman, R.P. de Groot, STAT3beta, a splice variant of transcription factor STAT3, is a dominant negative regulator of transcription, J. Biol. Chem. 271 (22) (1996) 13221e13227. C.C. Liang, A.Y. Park, J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro, Nat. Protoc. 2 (2) (2007) 329e333. A. Albini, Y. Iwamoto, H.K. Kleinman, G.R. Martin, S.A. Aaronson, J.M. Kozlowski, R.N. McEwan, A rapid in vitro assay for quantitating the invasive potential of tumor cells, Cancer Res. 47 (12) (1987) 3239e3245. H.D. Soule, T.M. Maloney, S.R. Wolman, W.D. Peterson Jr., R. Brenz, C.M. McGrath, J. Russo, R.J. Pauley, R.F. Jones, S.C. Brooks, Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10, Cancer Res. 50 (18) (1990) 6075e6086. P.J. Dawson, S.R. Wolman, L. Tait, G.H. Heppner, F.R. Miller, MCF10AT: a model for the evolution of cancer from proliferative breast disease, Am. J. Pathol. 148 (1) (1996) 313e319. S.J. Santner, P.J. Dawson, L. Tait, H.D. Soule, J. Eliason, A.N. Mohamed, S.R. Wolman, G.H. Heppner, F.R. Miller, Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells, Breast Canc. Res. Treat. 65 (2) (2001) 101e110. S.H. Kim, K. Zukowski, R.F. Novak, Rapamycin effects on mTOR signaling in benign, premalignant and malignant human breast epithelial cells, Anticancer
Res. 29 (4) (2009) 1143e1150. [16] J.C. van Deutekom, A.A. Janson, I.B. Ginjaar, W.S. Frankhuizen, A. Aartsma-Rus, M. Bremmer-Bout, J.T. den Dunnen, K. Koop, A.J. van der Kooi, N.M. Goemans, et al., Local dystrophin restoration with antisense oligonucleotide PRO051, N. Engl. J. Med. 357 (26) (2007) 2677e2686. [17] T. Williams, R. Kole, Analysis of prostate-specific membrane antigen splice variants in LNCap cells, Oligonucleotides 16 (2) (2006) 186e195. [18] M. Kinali, V. Arechavala-Gomeza, L. Feng, S. Cirak, D. Hunt, C. Adkin, M. Guglieri, E. Ashton, S. Abbs, P. Nihoyannopoulos, et al., Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study, Lancet Neurol. 8 (10) (2009) 918e928. [19] U.M. Wegenka, J. Buschmann, C. Lutticken, P.C. Heinrich, F. Horn, Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level, Mol. Cell Biol. 13 (1) (1993) 276e288. [20] L. Zhang, D.B. Badgwell, J.J. Bevers 3rd, K. Schlessinger, P.J. Murray, D.E. Levy, S.S. Watowich, IL-6 signaling via the STAT3/SOCS3 pathway: functional analysis of the conserved STAT3 N-domain, Mol. Cell. Biochem. 288 (1e2) (2006) 179e189. [21] S. Bhattacharya, R.M. Ray, L.R. Johnson, STAT3-mediated transcription of Bcl-2, Mcl-1 and c-IAP2 prevents apoptosis in polyamine-depleted cells, Biochem. J. 392 (Pt 2) (2005) 335e344. [22] Y. Yamanaka, K. Nakajima, T. Fukada, M. Hibi, T. Hirano, Differentiation and growth arrest signals are generated through the cytoplasmic region of gp130 that is essential for Stat3 activation, EMBO J. 15 (7) (1996) 1557e1565. [23] S. Dewilde, A. Vercelli, R. Chiarle, V. Poli, Of alphas and betas: distinct and overlapping functions of STAT3 isoforms, Front. Biosci. 13 (2008) 6501e6514.