Downregulation of YAP-dependent Nupr1 promotes tumor-repopulating cell growth in soft matrices

Despite decades of significant progress in understanding the molecular mechanisms of malignant tumorigenic cells, it remains elusive what these tumorigenic cells are and what controls the growth of these malignant cells. Recently, we have mechanically selected and grown highly malignant and tumorigenic tumor-repopulating cells (TRCs), a small sub-population of cancer cells, by culturing single cancer cells in soft fibrin matrices. However, it is unclear what regulates TRC growth besides Sox2. Here we show that nuclear protein 1 (Nupr1), a protein independent of Sox2, is downregulated in TRCs of melanoma, ovarian cancer and breast cancer cultured in soft fibrin matrices. Nupr1 expression depends on nuclear translocation of YAP that is enriched at the Nupr1 promoter sites; YAP is controlled by Cdc42-mediated F-actin and Lats1 interactions. Nupr1 regulates tumor-suppressor p53 and negatively regulates Nestin and Tert that are independent of Sox2 and promote TRC growth. Silencing Nupr1 increases TRC growth and Nupr1 overexpression inhibits TRC growth in culture and in immune-competent mice. Our results suggest that Nupr1 is a suppressor of growth of highly tumorigenic TRCs and may have a critical role in cancer progression.


INTRODUCTION
Despite decades of continuous efforts and significant progress in understanding the molecular mechanisms of malignant tumorigenic cells, 1 it remains unresolved what these tumorigenic cells are and what controls the growth and proliferation of these tumor cells. Recently, we mechanically selected tumorigenic tumor-repopulating cells (TRCs) independent of surface stem cell markers, a small sub-population of cancer cells, by culturing single cancer cells from cancer cell lines or primary tumors in three-dimensional (3D) soft fibrin matrices. 2 These TRCs exhibit highly tumorigenic and proliferative capacity and they efficiently repopulate tumors at distant organs in wild-type syngeneic and non-syngeneic mice. 2 The soft fibrin matrices promote TRC growth by facilitating Sox2 expression. 3 However, the melanoma cells can significantly increase their proliferation when Sox2 expression is only modestly increased, 3 suggesting that other factors that are regulated by matrix softness may contribute to TRC proliferation independent of Sox2. Using microarrays to screen for potential factors, we have identified nuclear protein 1 (Nupr1) that is markedly reduced in the TRCs in 3D soft fibrin matrices when compared with melanoma cells on 2D rigid plastic. Nupr1 is a DNA-binding protein implicated in regulation of cell cycle and apoptosis. 4 Nupr1 is also called COM1 (candidate of metastasis 1) because it is expressed in metastatic cancer cells. 5 The exact role of Nupr1 in cancer is poorly understood as several studies have associated Nupr1 with cancer cells' ability to survive [6][7][8][9] while another shows its tumor-suppressing capabilities. 10 Here we reveal that Nupr1 is downregulated in TRCs to facilitate proliferation of the tumorigenic cells and its expression depends on nuclear Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ).

Substrate stiffness regulates Sox2-independent Nupr1
To determine the impact of matrix rigidity on Nupr1, we compared its expression in cells that are cultured on 2D rigid plastic with that in cells in 3D soft fibrin matrices. Nupr1 mRNA expression was decreased by~70% in 3D soft (90-Pa) fibrin matrices ( Figure 1a) and Nupr1 protein levels were decreased considerably (Supplementary Figure 1a) when compared with that on 2D rigid plastic. Nupr1 expression was also much lower in human ovarian cancer cells (A2780) and human breast cancer cells (MCF-7) cultured in 3D soft fibrin gels (Supplementary Figure 1b), suggesting that low mRNA expression of Nupr1 is not limited to melanoma cells in 3D soft substrates. Increasing 3D matrix rigidity from 90 to 1050-Pa significantly upregulated Nupr1 expression ( Figure 1a). We define the melanoma cells that are selected and grown in 90-Pa fibrin gels for 5 days as TRCs. 3 Importantly, Nupr1 expression in the cells plated on 2D substrates increased by 47-fold when substrate stiffness was increased from 150-Pa to 8 kPa or rigid plastic (Figure 1b), suggesting that it is the substrate rigidity and not the substrate dimensionality that dictates Nupr1 gene expression. Silencing Nupr1 had no effect on Sox2 expression ( Figure 1c); silencing Sox2 had no effect on Nupr1 expression either ( Figure 1d). These data suggest that Nupr1 can be regulated by matrix rigidity and that Nupr1 is independent of Sox2.  ; n = 10 randomly chosen viewfields; ***P o0.001 (c) Western blotting for phosphorylated YAP S127 (p-YAP s127) and YAP in whole-cell lysates of melanoma cells after 3 days of culture on 2D rigid plastic or in 3D 90-Pa fibrin gels. (d) Real-time PCR of Ctgf and Cyr61 as indexes of YAP transcriptional activity on 2D and in 90-Pa 3D fibrin gels. Mean ± s.e.m.; n = 3; **P o0.01. and the nucleus, where they interact with transcriptional enhancer associate domain transcription factors to regulate transcription. YAP is known to sense cytoskeletal tension and mediate cellular mechanoresponses. 13 We assayed endogenous YAP subcellular localization by immunofluorescence for cells plated on 2D rigid plastic and in 3D 90-Pa substrates. Almost all YAP in cells on 2D rigid plastic was nuclear, whereas only~18% YAP in cells in 3D 90-Pa fibrin matrices was nuclear (Figures 2a and b). Furthermore, immunoblots show that 3D 90-Pa fibrin gels promoted YAP phosphorylation on serine 127 (ser 127 ), whereas the total amounts of YAP in 2D and in 3D 90-Pa were similar (Figure 2c), consistent with published results that phosphorylated YAP do not translocate to the nucleus. 14,15 Ctgf and Cyr61, two known YAP/TAZ endogenous targets, were used as markers of transcriptional activity of YAP. Ctgf and Cyr61 mRNA expression levels in cells in 3D 90-Pa were markedly reduced when compared with those on 2D (Figure 2d), supporting the finding that little YAP from TRCs (the cells in 3D 90-Pa) was translocated from the cytoplasm to the nucleus.
Cdc42 regulates Nupr1 via YAP As a key cytoskeleton regulator, Cdc42 is known to regulate the actin cytoskeleton to promote filopodia formation. 16,17 We have reported that Cdc42 is significantly downregulated in TRCs compared with control melanoma cells (the cells on 2D rigid plastic). 3 To investigate the relationship between Cdc42 and YAP, we knocked down Cdc42 in the cells on 2D rigid plastic. In comparison with the cells transfected with negative control small interfering RNA (siRNA), the cells transfected with Cdc42 siRNA exhibited dominant YAP cytoplasmic localization (Figures 3a and  b) and high levels of phosphorylation of YAP on serine 127 ( Figure 3c). To determine the role of nuclear YAP in Nupr1, we performed chromatin immunoprecipitation (ChIP) assay and found that YAP was enriched by 7-to 10-folds at Nupr1 promoter sites for melanoma cells on 2D rigid plastic (Figures 4a and b). For cells cultured in 3D 90-Pa fibrin gels, the YAP enrichment was reduced by~70% when compared with those on 2D rigid plastic (Supplementary Figure 2a), suggesting the specific role of YAP in Nupr1 gene expression. Silencing YAP downregulated Nupr1 expression but had no effect in Cdc42 expression ( Figure 4c Figure 5c). Latrunculin A had no effect on Cdc42 expression but decreased Nupr1 expression ( Figure 4c) consistent with published reports that F-actin interacts with Lats1, which in turn regulates phosphorylation of YAP. 14 Phosphorylated-YAP was higher in TRCs than in cells on rigid plastic and silencing Cdc42 elevated phosphorylated YAP (Supplementary Figure 6). In addition, silencing Cdc42 downregulated YAP activation markers Ctgf and Cyr61, and also Nupr1  Figure 7c). Together, these data suggest that Cdc42 that promotes assembly of F-actin and Lats1 binding to F-actin is upstream of YAP and regulates Nupr1 via YAP translocation.

Nupr1 is a negative regulator of melanoma cell growth in vitro and in vivo
To determine the functional role of Nupr1 in melanoma cells, we altered Nupr1 levels and assayed melanoma cell proliferation. Compared with the cells grown in 3D 90-Pa gels transfected with negative controls, silencing Nupr1 significantly increased colony size and colony number (Figures 5a and b; Supplementary Figures  8 and 9). In contrast, overexpressing Nupr1 markedly reduced colony size and number (Figures 5c and d; Supplementary Figures  8 and 9). These results suggest that Nupr1 negatively regulates TRC growth.
It is reported 8 that Nupr1 interacts with tumor-suppressor p53. p53 mRNA levels ( Figure 6a) and protein levels ( Figure 6b) were much lower in cells plated in 3D soft fibrin gels (TRCs) than in   (Figures 6a and b); silencing YAP and Cdc42 in cells on rigid plastic that are upstream of Nupr1 or treating cells on rigid plastic with Latrunculin A to disrupt F-actin significantly decreased p53 expression (Figure 6a). Together, these results suggest that Nupr1 is upstream of p53. Silencing p53 in cells on rigid plastic significantly increased colony size and colony number, consistently with the known role of p53 as a tumor suppressor (Figures 6c and d).
To further explore the role of Nupr1 in vivo, we either silenced Nupr1 in melanoma cells grown on 2D rigid plastic (whose Nupr1 levels were high; Figure 1a; Supplementary Figure 1a) or overexpressed Nupr1 in TRCs (whose Nupr1 levels were low;  From the published results on limiting dilution assays, it is known that 100 subcutaneously injected TRCs but not 100 melanoma cells grown on 2D rigid plastic can generate tumors; when 100 000 cells grown on rigid plastic are injected, however, 100% mice have tumors, 2 presumably a small population of the cells grown on rigid plastic are TRC-like cells. 2 Hence, we chose 2000 tumor cells to inject subcutaneously in order to determine the potential differences in tumorigenic efficiency (see Table 1) and tumor growth rates when Nupr1 was perturbed. (a) In all, 2000 cells cultured on 2D rigid plastic were transfected with negative control siRNA (Neg Ctr) or Nupr1 siRNAs (siNupr1) for 24 h and then subcutaneously injected to mice. The tumor volume was quantified using the formula of length times the square of width times 0.52. (b) Overexpressing Nupr1 suppresses tumor growth. 2000 TRCs transfected with empty vector (TRC+empty vector) or Nupr1 complementary DNA plasmids (TRC+Nupr1 overexpression) for 12 h were subcutaneously injected to mice. The volume of skin melanoma was measured for 25 days. Mean ± s.e.m.; n = 9 (Neg Ctr) and 12 (siNupr1) in a; n = 15 (empty vector) and 9 (Nupr1 overexpression) in b; data were pooled from two independent experiments as results from the two experiments were similar, in each experiment eight mice were used per condition; *P o0.05; **Po 0.01; ***Po 0.001. Welch's unpaired t-test.  (Table 1). Importantly, sizes of the tumors were significantly increased when Nupr1 was silenced and were significantly suppressed when Nupr1 was overexpressed (Figures 7a and b). These findings are consistent with the in vitro colony size results and support the notion that Nupr1 behaves like a tumorsuppressor in vivo.

Nupr1 negatively regulates nestin and Tert
To further explore what else may be regulated by Nupr1, we examined Nestin and Tert, which are upregulated in TRCs 2 (Figure 8a). Silencing Sox2 had no effects on either Nestin or Tert, suggesting these two molecules are independent of Sox2 (Figure 8b). Knocking down Nupr1, YAP or Cdc42, or disrupting F-actin with Latrunculin A, led to upregulation of Nestin and Tert (Supplementary Figure 8a), suggesting that Nupr1 and molecules that are upstream of Nupr1 negatively regulate Nestin and Tert. Importantly, silencing either Nestin or Tert decreased both colony size and colony number (Figures 8c and d), suggesting that Nestin and Tert facilitate cell growth. Together, these results suggest that Nestin and Tert are independent of Sox2 and dependent on Nupr1 and promote cell proliferation.

DISCUSSION
Our current reveals an important role of Nupr1 in inhibiting growth of tumorigenic cancer cells in vitro and in vivo and the pathway that is critical for Nupr1 expression. Published reports show that Sox2 is critical in pluripotency maintenance 18 of embryonic stem cells and fate determination 19,20 of adult stem cells. Sox2 upregulation has been found in tumor-initiating cells or precursors of osteosarcomas, 21 glioblastoma, 22 lung and esophageal squamous cell carcinomas, 23 and breast cancer. 24 In our previous studies, we have shown that Sox2 is markedly upregulated in melanoma TRCs. 2,3 More recently, we have discovered an essential role of Sox2 in mediating efficient extravasation of melanoma TRCs via downregulating Cdc42 and thus F-actin in a zebrafish model. 25 However, Sox2 may not be the only critical factor in various carcinomas as in many carcinomas there is no evidence that Sox2 is upregulated. Our current study reveals that Nupr1, independent of Sox2, is low in three types of carcinoma TRCs (melanoma, ovarian cancer and breast cancer) and is a negative regulator of TRC growth. We find that regulation of growth of TRCs by the soft mechanical microenvironment is complex. As the matrix stiffness becomes very low (~100 Pa), Cdc42 is lowered, which leads to decreases in F-actin, which in  Tumor-repopulating cell growth Q Jia et al turn frees up Lats1 so that it binds to and phosphorylate YAP, preventing translocation of YAP into the nucleus. As a result, Nupr1 is downregulated; downregulation of Nupr1, in turn, leads to downregulation of p53 and upregulation of Nestin and Tert. All these help to promote growth and self-renewal of TRCs (Supplementary Figure 10). This pathway appears to be independent of the Sox2-mediated pathway but is regulated by the soft matrices of the microenvironment. At the present, we do not know how important Nupr1 is in TRCs in many other carcinomas, but the abrogation of the inhibitory role of Nupr1 in soft matrices and the upregulation of Nupr1 in stiff matrices support the postulate of physical microenvironment barrier in cancer progression 26 and are consistent with the role of 3D stiff matrices in inhibiting carcinoma progression. 3 In the future, we need to determine how Nupr1, together with other tumor suppressors, inhibits carcinoma progression in human subjects and to find ways to perturb this pathway to stop cancer progression.

MATERIALS AND METHODS Animals
Four-week old C57BL/6 female mice were obtained from Center of Medical Experimental Animals of Hubei Province (Wuhan, China). The mice were randomly assigned to be in the control group or the treated group. As a minimum of six mice per group was required for having a statistical power, each group had eight mice, The experimentalists were blinded from the expected outcome of the treatment. 3D fibrin gel preparation Salmon fibrinogen and thrombin were purchased from Reagent Proteins (San Diego, CA, USA). 3D fibrin gels were prepared as described previously. 2,3 In brief, fibrinogen was diluted into 20 mg/ml with T7 buffer (pH 7.4, 50 mM Tris, 150 mM NaCl). Cells were detached from 2D rigid dishes. Fibrinogen and single-cell solution mixture was made by mixing the same volume of fibrinogen solution and cell solution, resulting in 1 or 8 mg/ml fibrin gels (the stiffness of 1 and 8 mg/ml fibrin gels is 90 and 1050 Pa, respectively). In all, 250 μl cell/fibrinogen mixture was seeded into each well of 24-well plate and mixed well with pre-added 5 μl thrombin (100 U/ml). The cell culture plate was then incubated in 37°C cell culture incubator for 25 min. Finally, 1 ml of minimum essential medium containing 10% fetal bovine serum and antibiotics was added.

F-actin staining
Cells were fixed with 4% paraformaldehyde for 10 min at room temperature. The F-actin was stained using 0.76 mM rhodaminephalloidin (Sigma, St Louis, MO, USA, 94072 Atto 565 phalloidin) and the nuclei was stained with 10 mg/ml 4,6-diamidino-2-phenylindole (Biosharp, BS130A) for 30 min at 37°C. The samples were rinsed three times with 1X phosphate-buffered saline before imaging. F-actin content was quantified along the lines shown using Image J (NIH, Bethesda, MA, USA).

ChIP assay
We performed ChIP assay following the manufacturer's instructions (EZ-ChIP kit, Millipore). Briefly, cells were subjected to cross-linking with 1% formaldehyde in medium for 10 min at 37°C and then lysed on ice. Chromatin was sonicated to shear DNA to an average length of 0.2-1.0 kb. ChIP was performed using control rabbit IgG or antibody against YAP (CST, #14074). The immunoprecipitation was heated to reverse the formaldehyde cross-linking and the DNA fragments in the precipitates was purified for real-time qPCR and PCR analysis. Primers were sets corresponding to Nupr1 and GAPDH (negative control) promoter regions. Colony number assay By changing the focal planes along the z axis (the direction of gel depth), the colony number was counted view by view. At least three wells of colonies were counted per condition per day.

Statistical analysis
All statistics were performed using a two-tailed Student's t-test with unequal variance except the Fisher's exact test and Welch's unpaired t-test for analyzing data from mice experiments.