FAM3C‐YY1 axis is essential for TGFβ‐promoted proliferation and migration of human breast cancer MDA‐MB‐231 cells via the activation of HSF1

Abstract Family with sequence similarity three member C (FAM3C) (interleukin‐like EMT inducer [ILEI]), heat shock factor 1 (HSF1) and Ying‐Yang 1 (YY1) have been independently reported to be involved in the pathogenesis of various cancers. However, whether they are coordinated to trigger the development of cancer remains unknown. This study determined the role and mechanism of YY1 and HSF1 in FAM3C‐induced proliferation and migration of breast cancer cells. In human MDA‐MB‐231 breast cancer cell line, transforming growth factor‐β (TGFβ) up‐regulated FAM3C, HSF1 and YY1 expressions. FAM3C overexpression promoted the proliferation and migration of MDA‐MB‐231 cells with YY1 and HSF1 up‐regulation, whereas FAM3C silencing exerted the opposite effects. FAM3C inhibition repressed TGFβ‐induced HSF1 activation, and proliferation and migration of breast cancer cells. YY1 was shown to directly activate HSF1 transcription to promote the proliferation and migration of breast cancer cells. YY1 silencing blunted FAM3C‐ and TGFβ‐triggered activation of HSF1‐Akt‐Cyclin D1 pathway, and proliferation and migration of breast cancer cells. Inhibition of HSF1 blocked TGFβ‐, FAM3C‐ and YY1‐induced proliferation and migration of breast cancer cells. YY1 and HSF1 had little effect on FAM3C expression. Similarly, inhibition of HSF1 also blunted FAM3C‐ and TGFβ‐promoted proliferation and migration of human breast cancer BT‐549 cells. In human breast cancer tissues, FAM3C, YY1 and HSF1 protein expressions were increased. In conclusion, FAM3C activated YY1‐HSF1 signalling axis to promote the proliferation and migration of breast cancer cells. Furthermore, novel FAM3C‐YY1‐HSF1 pathway plays an important role in TGFβ‐triggered proliferation and migration of human breast cancer MDA‐MB‐231 cells.


| INTRODUC TI ON
Breast cancer is one malignant tumour that occurs in the breast epithelium. It was reported that breast cancer had become the most common cancer in the female population worldwide in 2012. 1 In 2012, about 520 000 people died of breast cancer worldwide. 1 In China, breast cancer is the most common cancer and accounts for about 15% of newly diagnosed cancers in women. 2 It was estimated that there would be 2.5 million breast cancer patients in China by 2035. 2 Clearly, breast cancer has become a severe disease endangering the health of women worldwide. More intensive studies are needed to probe the mechanisms of breast cancer.
Transforming growth factor-β (TGFβ) protein has dual roles in the development of tumorigenesis. It inhibits tumour growth by inducing cell cycle arrest and apoptosis in the early stage of tumour formation. 3,4 However, with the progression of tumour, TGFβ stimulates the proliferation and migration of cancer cells, promoting tumour growth and invasion. 3,4 Protein kinase B (Akt) is an important intracellular molecule that stimulates cell proliferation and inhibits apoptosis 5,6 beyond its roles in regulating glucose and lipid metabolism as the key molecule of insulin signalling transduction. 7,8 It has been reported that Akt activation plays a critical role in TGFβ-promoted development of breast cancer. 9,10 Family with sequence similarity 3 (FAM3) cytokine-like gene family discovered in 2002 consists of four members designated as FAM3A, FAM3B, FAM3C and FAM3D, respectively. 13 It has been reported that FAM3C is involved in TGFβ-induced epithelial-mesenchymal transition (EMT), 14 and it is also called interleukin-like EMT inducer (ILEI).
Knockdown of FAM3C inhibits TGFβ-induced EMT. 14 So far, although FAM3C expression has been reported to be increased in various cancer tissues including breast cancer tissue, 14,15 its mechanism(s) in tumour cell proliferation and migration still remains largely unknown.
Particularly, the role of FAM3C in TGFβ-induced Akt activation and cell proliferation/migration in breast cancer cells remains unrevealed.
Heat shock factor 1 (HSF1) is a transcription factor controlling the process of stress, shock response, development and many other biological processes by inducing the expression of heat shock proteins (HSPs). 21,22 Heat shock factor 1 is also activated in breast cancer tissue, and its high expression level is associated with poor prognosis of breast cancer. 23 Beijing, China, the same concentration of scrambled siRNAs was used as negative control) for 12 hours, followed by the treatment with Ad-FAM3C or 2 ng/µL TGFβ for 12 hours. All siRNAs used in this study are naked siRNA, and the siRNA sequences were listed in Table S1.

| Western blotting assays
Proteins were extracted from cells using Roth lysis buffer containing fresh protease and phosphatase inhibitors (Applygen, Beijing, China). HSF1 protein expressions were increased. In conclusion, FAM3C activated YY1-HSF1 signalling axis to promote the proliferation and migration of breast cancer cells.
Furthermore, novel FAM3C-YY1-HSF1 pathway plays an important role in TGFβ-triggered proliferation and migration of human breast cancer MDA-MB-231 cells.

K E Y W O R D S
breast cancer, FAM3C, HSF1, YY1 Cell lysates were centrifuged at 13 680 g for 10 minutes at 4°C. Protein contents in the supernatant were quantified using bicinchoninic acid (BCA) Protein Assay Kit (Thermo scientific, Waltham, MA, USA). Protein samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was conducted using primary antibodies against target genes. After overnight incubation with primary antibodies, membranes were washed and incubated with horseradish peroxidaseconjugated secondary antibodies (Biodragon, Beijing, China) and then were detected using chemiluminescence kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analysed using a rabbit polyclonal as loading control. Anti-

| Real-time PCR assays
Total RNA (3-5 μg) isolated from cultured cells was converted to cDNA using cDNA synthesis kit (Thermo scientific) following the manufacturer's standard protocol. The protocol for real-time PCR analysis is as following: 95°C for 5 minutes, followed by 40 cycles at 95°C for 30 seconds, 59°C for 30 seconds and 72°C for 30 seconds. The Cycle threshold (Ct) values for the targets and GAPDH genes were provided by real-time PCR instrumentation. The comparative method 2 −ΔΔCt was used for the relative quantification of target gene transcription between the control and the treated groups. 27,28 All primer sequences for real-time PCR assays were listed in Table S2.

| Cell counting by haemocytometer
After treatments, the cells were split and resuspended in culture medium. The cell suspension was thoroughly mixed and the cells were

| Plasmid transfection
One day before transfection, an appropriate amount of MDA-MB-231 cells were seeded in a six-well plate. When the cells were about 70% confluence, they were transfected with HSF1 or YY1

| Cell migration assays
Cell motility was assessed using a wound healing assay. Treated cells were wounded by a 200 μL plastic pipette tip, and washed using phosphate-buffered saline (PBS) to remove cellular debris. After 0 and 12 hours, images of the wound areas under each condition were photographed. Migration rate was calculated by measuring the move distance of cells as detailed previously. 6 Moreover, the rate of wound healing was also evaluated by calculating wound area as detailed elsewhere 29,30 using ImageJ software (http://rsb.info.nih. gov/nih-image/). In the later method, the wound closure rate was determined using the formula: wound closure rate (%) = ([wound area 0h − wound area 12h ]/wound area 0h ) × 100.

| Transwell migration assay
The method for transwell migration assay was detailed elsewhere. 6 Representative images of cell density after FAM3C silencing. E, Cell number counting assays after FAM3C silencing. F, Cell viability assays after FAM3C silencing. N = 3-6, *P < 0.05 vs scramble-treated cells, #P < 0.05 vs scramble + TGFβ group of cells. FAM3C, family with sequence similarity three member C; HSF1, heat shock factor 1; TGFβ, transforming growth factor beta; Akt, protein kinase B stained with a crystal violet staining solution. The crystal violet was dissolved in 33% ethanoic acid, and the absorbance was determined at 570 nm. The data were normalized to the control value.

| Cell cycle detection assay
When the cells reached 60%-80% confluence, cells were treated with siRNA, Ad-FAM3C or TGFβ for 24 hours. The cells were collected and washed twice with PBS, fixed with 70% ethanol for 1 hour at 4°C, washed again with PBS, and re-suspended with propidium iodide (PI) solution (0.05 mg/mL) containing RNase. Next, they were incubated at room temperature in the dark for 30 minutes. The DNA content was analysed using a flow cytometer. The data in each read were normalized by the data of ranlila luciferase.

| Immunohistochemistry
Three breast cancer tissues and their corresponding adjacent normal tissues were stained with FAM3C, YY1 and HSF1 antibodies, respectively. For immunohistochemistry, sections were incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. Tissue

| Statistical analysis
The results are presented as the mean ± SEM. Statistical significance of differences between groups were analysed by t test. P values <0.05 were considered as statistically significant.

| FAM3C promoted the proliferation and migration of MDA-MB-231 cells concomitant with HSF1 up-regulation
Because TGFβ activation is highly associated with breast cancer, it  Figure S2A,B). siRNA

| Inhibition of HSF1 reversed the stimulatory effects OF FAM3C and TGFβ on the proliferation and migration of MDA-MB-231 cells
To further determine the roles of HSF1 in FAM3C-and TGFβ-promoted proliferation and migration of breast cancer cells, its activity was blocked using a selective inhibitor KRIBB11. Treatment with

| YY1 activated HSF1 to induce the proliferation of breast cancer cells
Because FAM3C overexpression or silencing is associated with HSF1 mRNA level change in breast cancer cells, it is reasonable to speculate that FAM3C enhanced HSF1 gene transcription. To identify the potential transcription factors that mediate FAM3C-induced transcription enhancement of HSF1 gene, the putative binding sites for some certain important transcription factors in human HSF1 gene promoter between −2000 bp and the transcription start site (0 bp) had been analysed using bioinformatic method. The potential binding sites with high prediction scores for certain transcription factors were indicated in Figure S6. As a result, bioinformatic prediction revealed that six putative binding sites highly specific for transcription factor YY1 were existed in the promoter region of human HSF1 gene ( Figure   S6). Reference mining revealed that YY1 deregulation had been reported to be involved in the pathogenesis of breast cancer. 33 Figure 5H). Overall, these findings revealed that HSF1 is a direct target gene of YY1, which stimulated the proliferation and migration of breast cancer cells by inducing HSF1 expression.

| FAM3C and TGFβ promoted the proliferation and migration of breast cancer cells via YY1-HSF1 pathway
To further determine the role of YY1 in FAM3C-induced HSF1 upregulation, and the proliferation and migration of breast cancer cells,

| FAM3C-YY1-HSF1 signalling axis is activated in human breast cancer tissues
To validate the roles of FAM3C-YY1-HSF1 signalling axis in the pathogenesis of breast cancer, FAM3C, YY1 and HSF1 protein expressions were analysed in human breast cancer tissues. Immunohistochemical staining revealed the protein levels of FAM3C, YY1 and HSF1 were increased in human breast cancer tissues when compared with adjacent normal tissues ( Figure 8A-C, Figure S14A-C).

| D ISCUSS I ON
The transcription factor YY1 was discovered in the 1990s 35,36 . YY1 belongs to the GLI-Kruppel zinc finger transcription factor family, and has many biological functions by regulating the expressions of its target genes. 37 It has been estimated that YY1 may regulate about 10% of mammalian gene expressions. 38,39 Recently, there had been increasing evidence that YY1 also plays important roles in the pathogenesis of various cancers including breast cancer. 40,41 In human breast cancer tissues, YY1 expression is increased, and high expression of YY1 protein level is associated with poor prognosis of breast cancer in human. 33,34,40,41 However, Lea MH et al also reported that YY1 protein level is lower in the breast cancer tissues of breast cancer patients than in the normal breast tissues. 33 Moreover, YY1 overexpression represses the proliferation and migration of breast cancer cell lines. 33 Clearly, more intensive studies are needed to clarify the role and mechanism of YY1 in breast cancer, in particular, at the different stage of tumour development.
FAM3C, 14,15 HSF1 23,24 and YY1 33,34 have been independently reported to be associated with breast cancer, and all of them could serve as potential diagnostic biomarkers and therapeutical targets. However, whether they are coordinated to promote the development of cancers remains unknown. This study revealed that HSF1 is a direct target gene of YY1, which induces HSF1 gene transcription. FAM3C up-regulates YY1 to induce HSF1 expression, finally activating Akt-Cyclin D1 pathway to promote the proliferation and migration of breast cancer cells. For the first time, our findings revealed that these important molecules are coordinated to promote the proliferation and migration of breast cancer cells. Clearly, the roles of FAM3C, YY1 and HSF1 in the pathogenesis of breast cancer and/or other cancers should be considered as a whole. The novel FAM3C-YY1-HSF1 signalling axis also provides a new explanation mechanism for TGFβrelated breast cancer. An increase in FAM3C expression due to excessive TGFβ production plays important roles in inducing YY1 and HSF1 expressions as observed in human breast cancer or other cancer tissues. In one previous study, Chaudhury A et al found that TGFβ-induced EMT is associated with the up-regulation of FAM3C and activation of Akt. 44 However, the relationship between FAM3C up-regulation and Akt activation remained unanswered in the same study. 44 The current and our previous findings 27,28 revealed that FAM3C likely mediates TGFβ-promoted Akt activation in the pathogenesis of EMT through YY1-HSF1 signalling axis. New FAM3C-YY1 pathway also provides an explanation for the findings that TGFβ induces HSF1 expression with unclear mechanism in ovarian and breast cancer cells. 45,46 Interestingly, although YY1-HSF1 pathway plays an important F I G U R E 8 FAM3C-YY1-HSF1 axis was activated in human breast cancer tissues. (A-C) Immunohistochemical staining revealed that FAM3C (A), YY1 (B) and HSF1 (C) protein levels were increased in breast cancer tissues when compared normal breast tissues. Three breast cancer tissues and their corresponding adjacent normal tissues were stained with FAM3C, YY1 and HSF1 antibodies, respectively. The amplification power had been marked in the images. The cells with positive staining by the indicated antibodies exhibited brown, and representative positive cells had been marked by arrows in the images. D, Proposed mode of FAM3C-YY1-HSF1 signalling axis in the pathogenesis of breast cancer. FAM3C-YY1-HSF1 signalling axis is essential for TGFβ-promoted proliferation and migration of breast cancer cells. FAM3C, family with sequence similarity three member C; YY1, Ying-Yang 1; HSF1, heat shock factor 1; TGFβ, transforming growth factor beta; Akt, protein kinase B role in activating Akt, it has also been reported that Akt activation also activates YY1 and HSF1 expressions. 47,48 TGFβ activated FAM3C-YY1-HSF1 axis to promote proliferation and migration in various breast cancer cell lines. Collectively, these findings together suggested that FAM3C likely initiates the crosstalks among YY1, HSF1 and Akt, finally causing excessive Akt activation to trigger tumour growth and invasion.
Given that Akt plays essential roles in regulating metabolism 7 and promoting cancers, 49 FAM3C-YY1-HSF1 signalling axis may play unique role in maintaining the balance between metabolism and cancers by modulating Akt activity. Under insulin resistance, repression of FAM3C by the factors such as fatty acids will impair YY1-HSF1-Akt pathway to enhance hepatic gluconeogenesis and lipogenesis, exaggerating hyperglycaemia and fatty liver. 27,28 However, in case of TGFβ overproduction, long-term activation of FAM3C-YY1-HSF1 pathway will trigger the development of cancers in various tissues including breast tissue by promoting excessive Akt activation. Given the important roles of HSF1 in regulating hepatic glucose and lipid metabolism by modulating Akt activity and molecular chaperone expressions, 27,28,50,51 the side effects such as hyperglycaemia should be taken into consideration when HSF1 inhibitor is potentially used to treat cancers.
So far, the mechanism of FAM3C-induced YY1 activation still needs further exploration. Moreover, because breast cancer cells had been previously shown to secrete FAM3C protein, 32 it is of great significance to determine the association between circulating FAM3C protein and breast cancer progression in future. At present, it is difficult in accurately detecting the FAM3C protein level in the circulation due to the unavailability of high sensitive methods such as ELISA and RIA. Clearly, developing high sensitive methods for the determination of circulating FAM3C protein level will shed light on the diagnosis and treatment of breast cancer.
In summary, this study revealed that FAM3C, YY1 and HSF1 are coordinated to promote the proliferation and migration of human breast cancer MDA-MB-231 cells. FAM3C activates YY1 to induce HSF1 expression, which finally triggers the proliferation and migration of breast cancer cells by activating Akt. Furthermore, FAM3C-YY1-HSF1 signalling axis is essential for TGFβ-promoted proliferation and migration of human breast cancer cells ( Figure 8D).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R S ' CO NTR I B UTI O N S
WY, BF and YM researched data and contributed to discussion. WY and JW provided the technical assistance. WY, BF and YM wrote the manuscript. BF, YM, BG, QC and JY revised/edited manuscript. HZ, YY and JY designed the study and revised the manuscript. HZ provided human tissue slides. Dr JY is the guarantor of this work, and have full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the manuscript.