- Open Access
SALL1 functions as a tumor suppressor in breast cancer by regulating cancer cell senescence and metastasis through the NuRD complex
- Chunling Ma†1, 2,
- Fang Wang†1, 3,
- Bing Han†1, 4,
- Xiaoli Zhong1,
- Fusheng Si1,
- Jian Ye1,
- Eddy C. Hsueh5,
- Lynn Robbins6, 7,
- Susan M. Kiefer1,
- Yanping Zhang5,
- Pamela Hunborg5,
- Mark A. Varvares8, 9,
- Michael Rauchman6, 7Email author and
- Guangyong Peng1Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 21 January 2018
- Accepted: 11 March 2018
- Published: 6 April 2018
SALL1 is a multi-zinc finger transcription factor that regulates organogenesis and stem cell development, but the role of SALL1 in tumor biology and tumorigenesis remains largely unknown.
We analyzed SALL1 expression levels in human and murine breast cancer cells as well as cancer tissues from different types of breast cancer patients. Using both in vitro co-culture system and in vivo breast tumor models, we investigated how SALL1 expression in breast cancer cells affects tumor cell growth and proliferation, metastasis, and cell fate. Using the gain-of function and loss-of-function strategies, we dissected the molecular mechanism responsible for SALL1 tumor suppressor functions.
We demonstrated that SALL1 functions as a tumor suppressor in breast cancer, which is significantly down-regulated in the basal like breast cancer and in estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2) triple negative breast cancer patients. SALL1 expression in human and murine breast cancer cells inhibited cancer cell growth and proliferation, metastasis, and promoted cell cycle arrest. Knockdown of SALL1 in breast cancer cells promoted cancer cell growth, proliferation, and colony formation. Our studies revealed that tumor suppression was mediated by recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) complex by SALL1, which promoted cancer cell senescence. We further demonstrated that the mechanism of inhibition of breast cancer cell growth and invasion by SALL1-NuRD depends on the p38 MAPK, ERK1/2, and mTOR signaling pathways.
Our studies indicate that the developmental control gene SALL1 plays a critical role in tumor suppression by recruiting the NuRD complex and thereby inducing cell senescence in breast cancer cells.
- Tumor suppressor gene
- NuRD complex
- Breast cancer
The human SALL gene family, SALL1-SALL4, was identified as homologues to the Drosophila homeotic gene spalt [1–3]. Originally, the SALL family are zinc finger transcription factors that were shown to function as critical regulators in the development of multiple mammalian organs, including kidney, heart, and the hematopoietic system [4–6]. Mutations in the human SALL1 and SALL4 genes result in Townes-Brocks (TBS) and Okihiro syndrome (OS), respectively [1, 5, 7]. The SALL gene family is also important for the control of stem cell pluripotency, differentiation and self-renewal properties involving transcriptional and epigenetic actions [6, 8–10].
Besides the regulation of organ and stem cell development, the role of SALL genes in tumor biology and tumorigenesis has been recently investigated. SALL2 has been reported as a potential tumor suppressor in ovarian cancer and Wilms tumor [11–13]. SALL4 was shown to regulate survival and apoptosis in human leukemic cells [14, 15]. Furthermore, SALL4 was recently identified as a novel marker for hepatoblastoma, non-small cell lung carcinoma, and gastric cancinoma [16, 17]. Mutations in SALL3 have been discovered in a significant proportion of Burkitt’s lymphoma cases . It has been shown that the SALL1 promoter was methylated in breast and other epithelial cancers , but little is known about the role of SALL1 in the pathogenesis of human cancers. A recent report identified SALL1 as a tumor suppressor in human breast cancer, using an in vivo RNAi screen strategy . However, the molecular mechanism and causative role of SALL1 in the regulation of breast cancer development and tumorigenesis are not well understood.
The role of SALL1 in the regulation of organogenesis of the kidney has been extensively studied by our group and others. We have demonstrated that SALL1 recruits and binds to the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex and their combined action is required to maintain renal progenitor cells [4, 6, 21–23]. We identified a highly conserved 12-amino acid motif in the SALL1 that is sufficient for the recruitment of NuRD . We showed that protein kinase C phosphorylates serine 2 of SALL1 repression motif to regulate SALL1-mediated NuRD recruitment and its associated functions . Importantly, increasing evidence suggests that the NuRD protein complex plays an essential role in cancer development and metastasis . Specifically, several subunits of NuRD, such as MTA1, MTA3, and Mi-2 can directly control the cancer invasive growth, epithelial-to-mesenchymal transition, and metastasis in breast cancer [24–26]. Given the recent study identifying that SALL1 could be a tumor suppressor in human breast cancer , it is important to determine how SALL1 regulates breast cancer cell biology and functions. In addition, whether SALL1 recruits the NuRD complex to perform its tumor suppressor function in breast cancer is unclear. Improved understanding of these molecular processes mediated by SALL1 for the regulation of tumor biology and tumorigenesis will open new avenues to develop novel therapeutic strategies in human breast cancer and possibly other tumors.
To better understand the role of SALL1 in the pathogenesis of breast cancer, we investigated the mechanism of SALL1 tumor suppressor activity in breast cancer models. Using both gain-of function and loss-of-function strategies, we showed that SALL1 expression in breast cancer cells inhibited tumor cell growth and proliferation, promoted cell cycle arrest, and induced cell senescence. We further revealed that SALL1 tumor suppressor activity depended on its ability to recruit NuRD and that this molecular process was controlled by MAPK p38 and ERK1/2, and mTOR signaling pathways in cancer cells. In addition, our complementary in vivo studies demonstrated that SALL1 expression and NuRD recruitment in breast tumor cells inhibited tumorigenesis and metastasis in breast cancer models in vivo. Collectively, these studies suggest that SALL1 functions as a tumor suppressor in breast cancer and directly controls cancer cell fate and metastasis.
SALL1 expression is down-regulated in human breast cancer cell lines and tissues
SALL1 over-expression in breast cancer cells inhibits tumor cell growth and proliferation, and promotes cell cycle arrest
Suppression of tumor cell proliferation and growth mediated by SALL1 expression could be due to the induction of apoptosis or cytolysis in the tumor cells. We therefore measured apoptosis and cell death in SALL1-transfeced breast tumor cells. We found that breast tumor cells MCF7, MDA-MB-231 and E0771 in medium alone or transfected with control vector contained some apoptotic cells (around 10% in MCF-7, 4% in MDA, and 20% in E0771). However, overexpression of SALL1 in cancer cells did not induce increased apoptosis or cell death in breast cancer cell lines (Fig. 2c and Additional file 1: Figure S2C). In parallel, we studied the cell cycle distribution of the breast cancer cells transfected with SALL1. SALL1 transfection in MCF-7, MDA and E0771 cells significantly induced cancer cells to arrest in S phase and decrease in G0/G1 phase (Fig. 2d). Notably, transfection of SALL1 in melanoma B16F0 cells induced neither cell apoptosis nor cell cycle arrest. To further identify the potential mechanism responsible for the SALL1-mediated breast cancer cell arrest in S phase, we determined the cell cycle regulation gene expressions in MDA-MB-231 cells using Real-time PCR analysis, including Cyclin A2, B1, D1 and E1, as well as CDK2, 4 and 6. We observed that transfection with SALL1 significantly increased the gene expressions of Cyclin A2, Cyclin B1, Cyclin E1, CDK2 and CDK4 in breast cancer MDA cells, which are important for checkpoint regulation in G1-S transition and S phases (Additional file 1: Figure S3). These data suggest that over-expression of SALL1 in breast cancer strongly suppresses tumor growth and proliferation, as well as induces cell cycle arrest, which is mechanistically independent of apoptosis or cytolysis in tumor cells.
Knockdown of SALL1 in breast cancer cells promotes tumor cell growth, proliferation, and colony formation
SALL1 over-expression in breast cancer cells induces tumor cell senescence
The induction of DNA damage is the key molecular process in senescent cells, which could be induced by telomere erosion and/or other forms of stress. The nuclear kinase ataxia-telangiectasia mutated protein (ATM) is the chief inducer of the DNA-damage response. We thus determined whether induction of ATM-associated DNA damage is the main trigger for SALL1-induced senescence in breast tumor cells [33, 34]. Over-expression of SALL1 significantly induced active, phosphorylated ATM in MCF-7, MDA and E0771 cancer cells (Fig. 4c and Additional file 1: Figure S4B). In addition, we further investigated the other key DNA damage response proteins involved in the induction of senescence due to the DNA damage response. These proteins include ATM substrates H2AX and 53BP1, as well as the downstream target checkpoint kinase 2 (CHK2) [30, 34]. We observed that transfection of SALL1, but not SALL4 or control vector also significantly induced phosphorylation of H2AX, 53BP1 and CHK2 in MCF-7, MDA and E0771 cells (Data not shown). To confirm the involvement of ATM-associated DNA damage response in SALL1-mediated breast cancer cell senescence, we next determined whether we can prevent the SALL1-mediated senescence in breast cancer cells through the functional blockade of ATM-induced DNA damage using loss-of-function approaches with the specific pharmacological ATM inhibitor KU55933 and shRNA against ATM. As shown in Fig. 4d, treatment of MCF-7, MDA and E0771 breast cancer cells with KU55933 dramatically suppressed the phosphorylation of ATM in SALL1-transfected tumor cells and prevented induction of senescence in tumor cells. In addition, knockdown of ATM expression with shRNA significantly decreased the senescent cell populations in SALL1-transfected breast tumor cells, further confirming the involvement of the ATM-associated DNA damage response in SALL1-induced tumor cell senescence (Fig. 4e). These data provide the first evidence that suppression of breast cancer growth and proliferation mediated by SALL1 expression is due to the induction of tumor cell senescence.
SALL1 recruits NuRD in breast cancer performing a tumor suppressor function
In our efforts to identify the relationship and direct interactions between SALL1 and NuRD, we have demonstrated that protein kinase C phosphorylates serine 2 of the SALL1 repression motif and regulates the association with NuRD . Furthermore, we showed that substitution of the serine with a glutamic acid (SALL1-S2E, phosphomimetic) significantly abolished the effect on NuRD recruitment and repression activity; whereas mutating the serine to an alanine (SALL1-S2A) modestly increased the transcriptional repression [21, 22] (Additional file 1: Figure S5). We next utilized SALL1 constructs with these two separation-of-function mutations to test its effects on senescence induction in breast cancer cells. As we expected, transfection of SALL1-S2E into MCF-7 and E0771 breast cancer cells lost the ability to induce tumor cell senescence. In contrast, transfection of SALL1-S2A into tumor cells significantly augmented senescence induction in both cell lines compared with that of wild type SALL1-transfected tumor cells (Fig. 5c and d). To confirm the physical interaction of SALL1 with the NuRD complex in cancer cells, we transfected MCF-7 breast cancer cells with GST fusions of wild type SALL1, or SALL1-S2A and SALL1-S2E. GST-SALL1 fusion proteins were isolated on glutathione-Sepharose beads. Western-blot assays were then performed to determine the expression of components of the NuRD complex, including MTA2, RbAp46/48, HDAC1 and MBD3, after SALL1 and GST pulldown [21, 22]. Transfection of the three fusion constructs equivalently expressed SALL1 protein (Fig. 5e). Expression of wild type SALL1 and SALL1-S2A in MCF-7 tumor cells recruited the endogenous NuRD complex components. However, GST-SALL-S2E did not pulldown NuRD components even though it is expressed at a level comparable to wild type SALL1. Collectively, these results clearly indicate that SALL1 recruits NuRD in breast cancer cells resulting in suppression of tumor growth and proliferation, and induction of tumor cell senescence.
SALL1 induces selective modulation of MAPK p38 and ERK1/2, and mTOR signaling pathways in breast cancer cells
SALL proteins heterodimerize and in some contexts can cross-regulate their respective expression. We thus determined whether SALL1 could alter the expression of other members in the SALL family mediating breast cancer growth suppression . Our results showed that transfection of SALL1 in both MCF-7 and E0771 cells did not change the gene expression levels of SALL2, SALL3 and SALL4 at different time points using Real-time PCR analyses (Additional file 1: Figure S6).
In addition to MAPK signaling, mTOR kinase signaling activation is important for tumor cell proliferation and senescence induction [37–41]. We next investigated whether mTOR signaling is also involved in the SALL1-induced breast cancer growth inhibition and senescence induction. We determined the activation of mTOR and its downstream substrates p70S6K and 4E-BP1, in breast tumor cells after transfection with SALL1 . Transfection of SALL1 but not mutated SALL1 in both MCF-7 and E0771 breast tumor cells significantly induced the phosphorylation of mTOR, p70S6K, and 4E-BP1, further confirming the activation of mTOR signaling in tumor cells after SALL1 expression (Fig. 6d). Using loss-of-function strategies, we demonstrated that the mTOR inhibitor rapamycin and shRNA to specifically knock down mTOR gene expression in breast cancer cells dramatically prevented induction of senescence in tumor cells mediated by SALL1 expression (Fig. 6e and f). These results suggest that the mTOR signaling pathway is also critical in regulating breast cancer cell senescence mediated by SALL1 expression. Furthermore, our studies indicate that SALL1 expression in breast cancer selectively utilizes both MAPK and mTOR signaling pathways controlling tumor cell fate and functions.
SALL1 expression in breast cancer cells inhibits tumorigenesis and metastasis in vivo
SALL2 and SALL4 have been recently recognized as regulators of tumorigenesis [12–15], but little information is known about the role of the SALL1 gene in regulation of tumor biology. In the current study, we showed that SALL1 expression was significantly down-regulated in specific human breast cancer subtypes based on analyses of clinical tumor samples and cell lines. We further demonstrated that SALL1 expression in human and murine breast cancer cells controlled tumor cell growth and proliferation in vitro, and that overexpression of SALL1 inhibited tumorigenesis and metastasis in vivo in breast cancer xenograft models. Importantly, the tumor suppressor function mediated by SALL1 is mechanistically related to cell senescence induction via the recruitment of the NuRD complex in cancer cells. Our studies clearly indicate that SALL1 functions as a tumor suppressor in breast cancer, which could be a novel target for human breast cancer therapy.
A recent paper demonstrated that SALL1 could be a tumor suppressor in human breast cancer, using an in vivo RNAi screen strategy . They further showed that high expression of SALL1 was associated with significantly increased relapse-free survival, overall survival, metastasis-free survival, and tumor-free survival of breast cancer patients. However, whether and how SALL1 regulates human breast cancer is still unclear. Improved understanding of the molecular events should open new avenues for breast cancer clinical therapy. Our group has extensively studied the molecular mechinasms responsible for SALL1-mediated regulation in kidney development, and demonstrated that endogenous SALL1 recruits and binds to the NuRD complex to regulate transcriptional repression and specific developmental processes, such as progenitor cell fate [4, 6, 21–23]. In this study, using both the loss-of-function (either deletion of the conserved NuRD-binding 12-amino acid peptide motif or substitution of the serine with a glutamic acid SALL1), and gain-of-function (mutating the serine to an alanine) strategies in vitro and in vivo studies, we clearly demonstrated that SALL1 also utilizes a similar mechanism as in the developing kidney to recruit the NuRD complex, resulting in the inhibition of tumorigenesis and metastasis in breast cancer [21, 22]. In support of our novel finding, studies from other groups have already shown that the key components of NuRD complex, including MTA1, MTA3, and Mi-2 (CHD4), and other NuRD interacting proteins such as LSD1, directly control the invasive growth, epithelial-to-mesenchymal transition, and metastasis in breast cancer [24–26]. Furthermore, two groups identified frequent somatic mutations in the NuRD component chromodomain helicase DNA-binding protein 4 (CHD4) in an aggressive form of uterine cancer [46, 47]. Importantly, a more recent study demonstrated that loss of CHD4 leads to therapeutic resistance in BRCA2 mutant ovarian cancer . Our current work further suggests a causative link between SALL1 gene regulation, NuRD complex function, and breast cancer pathogenesis. In addition to the recruitment of the NuRD complex, SALL1 may also be involved in the regulation of other oncogenes, such as PTEN and c-Myc [10, 49]. However, we did not observe changes of these two oncogenes in breast cancer cells mediated by SALL1 over-expression (Data not shown). Notably, the MTA1, MTA2 and MTA3 components of the NuRD complex have been shown to play an important role in the ER and HER2 pathways regulating the epithelial-mesenchymal transition (EMT) and tumor cell invasion and metastasis, but they have distinct effects [24, 26]. Given that ER, PR and HER2 expression levels in tumor cells are important prognostic factors for breast cancer outcomes, we also determined whether SALL1 had different expression in breast cancer patients with different ER, PR or HER2 expression statuses. Our results suggested that SALL1 expression in HER2+ patients was much higher than that in HER2− patients. In addition, SALL1+ cell numbers in ER− patients were significant lower than those in ER+ patients. Notably, analyses of TCGA normalized log2 transformed breast cancer argilent microarray expression data sets clearly showed a significant decrease of SALL1 gene expression in the basal like breast cancer, as well as in ER−, PR− and triple negative breast cancer tissues. To further identify potential mechanism responsible for the down-regulation of SALL1 in breast cancer, we explored promoter methylation status of the SALL1 gene via COSMIC genome browser . Consistent with the previous report showing that the SALL1 promoter was methylated in breast and other epithelial cancers , our analysis also demonstrated that the genes were highly methylated in the 2 regions of the SALL1 promoter in the breast cancer tissues. In addition, studies from other groups have demonstrated that SALL1, which is located at 16q12.1 is a region that was shown to undergo loss of heterozygosity (LOH) in breast, prostate, ovarian cancers and in retinoblastoma [50, 51]. We will continue our efforts to identify the molecular interactions and regulatory mechanisms between SALL1, NuRD, and ER, PR and HER2, in the regulation of tumorigenesis and metastasis in breast cancer. In addition, given the multiple functions mediated by different subunits of the NuRD complex, identification of the precise assembly of NuRD components recruited by SALL1 in breast cancer cells will facilitate our understanding the functional role of SALL1 gene in tumor biology.
Besides the recruitment of NuRD complex, our current study also identified senescence induction as a novel mechanism mediated by SALL1 for the regulation of tumor biology and tumorigenesis in breast cancer. Cellular senescence was initially described more than 50 years ago in human fibroblasts with limited passages in cell culture. It is now well known that senescent cells have permanent cell cycle arrest, but remain viable, metabolically active and possess unique transcriptional profiles and gene regulation signatures. There are two major categories of cellular senescence: (1) Replicative senescence (telomere-dependent senescence) occurs due to telomere shortening or dysfunction that triggers a classical DNA-damage response [29, 30]; and (2) Premature senescence (extrinsic senescence or telomere-independent senescence) is induced by a variety of extrinsic forms of stress, such as oxidative stress, DNA damage, and activation of certain oncogenes, as well as some inflammatory cytokines and chemokines . We have recently demonstrated that human Treg cells and tumor cells can also induce ATM-associated DNA damage in responder T cells resulting in T cell senescence [31, 32, 34, 53]. In addition, cellular senescence is now thought to be a tumor suppressive mechanism that could be a harnessed as a possible cancer therapy strategy . In this study, we were the first to show that SALL1 also plays a critical role in control of genome stability, cell-cycle progression and cell fate in breast cancer. Specifically, we observed that SALL1 gene expression in breast cancer strongly suppresses tumor growth and proliferation, as well as induces cell cycle S phase arrest, which is mechanistically independent of apoptosis or cytolysis. We further discovered that SALL1-mediated suppression of breast cancer cells is due to the induction of tumor cell senescence as shown by induction of SA-β-Gal [31, 32, 53]. We identified that ATM-associated DNA damage is responsible for SALL1-mediated breast cancer cell senescence, by analyzing activation of ATM and its related targets, as well as using loss-of-function approaches with a specific pharmacological ATM inhibitor and shRNA. Importantly, we also provide evidence demonstrating that the SALL1-mediated suppression of tumor growth, cell proliferation and induction of tumor cell senescence depends on the endogenous recruitment of NuRD complex in breast cancer cells. The regulation of cell cycle transition and DNA damage responses mediated by the NuRD complex has been well recognized [24, 26, 54]. MTA1 and MTA2 can directly regulate p53 stability and function, leading to growth arrest inhibition and DNA damage response regulation . CHD4 is also as an important regulator of the G1/S cell cycle transition and ATM-associated DNA damage responses . In addition, HDAC1 and HDAC2 regulate the DNA-damage response and cellular senescence . Our previous studies have shown that SALL1 binding with NuRD directly repressed Gbx2, suggesting that Gbx2 is a direct SALL1 target gene [21, 22]. Furthermore, mutating the NuRD binding motif in SALL1 not only prevented binding of NuRD components, but the associated HDAC activity was also completely lost [21, 22]. Gbx2 was shown to be a marker of chemoresistance in triple negative breast cancer . However, how and whether Gbx2 and HDAC involve SALL1-mediated tumor cell DNA damage and senescence is still unknown in the current study. Future studies will continue to focus on the identification of the subunits of NuRD and target genes recruited by SALL1 in breast cancer cells responsible for the regulation of DNA-damage response and senescence induction. Interestingly, one study suggested that SALL2 directly binds to the p21 promoter promoting cell cycle arrest and inhibiting cell growth . SALL1 binds the p21 promoter and represses luciferase activity driven by this promoter in a NuRD dependent manner (Our unpublished observations). Consistent with this finding, it has been shown that CHD4 also binds the p21 promoter and inhibits expression of this cell cycle gene . Therefore, the ability of SALL1 to directly modulate cell cycle regulatory molecules, such as p21, is another potential mechanism that needs to be explored.
Dissection of the unique molecular signaling responsible for SALL1-mediated tumor suppression is another challenge. Our studies clearly showed that SALL1 expression in breast tumor cells selectively modulated the MAPK p38 and ERK1/2, as well as mTOR signaling pathways in tumor cells. In addition, the loss-of-function studies with specific pharmacological inhibitors and lentivirus-based shRNAs further indicated that SALL1-mediaed tumor suppression and senescence induction is controlled by both MAPK and mTOR signaling pathways. It is well recognized that MAPK signaling pathways play a major role in regulating cell cycle re-entry, oncogenic ras-induced senescence and G1 cell cycle arrest [35, 36]. Our recent studies further demonstrated that MAPK ERK1/2 and p38 signaling controls the molecular process of human CD4+CD25hiFoxP3+ Treg-induced responder T cell senescence . In addition to MAPK signaling, mTOR kinase signaling activation is important for tumor cell proliferation and senescence induction . mTOR signaling is also involved in the oncogene-induced DNA damage responses and cell senescence [39–41]. Our current studies further identified important roles of these two signaling pathways in SALL1-mediated regulation in breast cancer cells. However, the results presented here are different from our previous observations showing that SALL1 induces Wnt signaling in the developing kidney . Interestingly, we did not find activation of Wnt signaling in breast cancer MCF-7 and E0771 cells induced by SALL1 over-expression. These results suggest that the molecular signaling utilized by SALL1 promoting its tumor suppressor function is different from that in the regulation of organ development. Further dissection of how MAPK signaling and mTOR signaling cooperate and identification of unique adaptor molecules controlling SALL1 biological functions in tumor cells will be critical preludes for the application of SALL1 and tumor senescence as new targets for tumor therapeutic interventions.
We identified SALL1 as a novel tumor suppressor in breast cancer. We demonstrated that SALL1 can induce tumor cell senescence as a novel mechanism of tumor suppressor function. This molecular process acts through NuRD recruitment and is controlled by the MAPK and mTOR signaling pathways. These studies not only reveal a novel role of SALL1 in breast cancer biology, but also provide the mechanistic and causative links among SALL1 regulation, cellular senescence, NuRD, as well as MAPK and mTOR signaling pathways. These important aspects should provide new insights relevant for the development of novel therapeutic strategies in human breast cancer and other cancers as well.
Human samples and cell lines
Tumor samples were obtained from breast cancer patients treated at the Department of Surgery, Saint Louis University from 2004 to 2015 who have given informed consents for enrollment in a prospective tumor procurement protocol approved by the Saint Louis University Institutional Review Board. Paired fresh tumor tissues and normal breast tissues were obtained perioperatively and snap frozen in liquid nitrogen. In addition, fresh-frozen metastatic cutaneous melanoma tumor tissues were also collected as controls for this study. Breast tumor cell lines (human MDA-MB-231, MCF7, BC80, 31, 30, 29, 16, 12, 10, and murine 4 T1 and E0771), Melanoma cell lines (Mel1938, Mel1586, Mel1860, Mel1363, Mel1526 and Mel1628, and murine B16F0), prostate cell line PC3 and DU145, colon cancer cell line SW480 and lymphoma L428 and L504, as well as normal breast cells and fibroblast cells, were either obtained from the American Tissue Culture Collection (ATCC) or established by our group, and maintained in RPMI 1640 medium containing 10% fetal calf serum (FCS) and penicillin-streptomycin (Invitrogen, Inc. San Diego, CA).
Full length flu-tagged SALL1 wild type and mutant constructs cloned into pcDNA3.1 were prepared as previously described . Point mutants were created by PCR-mediated site directed mutagenesis using QuikChange (Stratagene). The amplified PCR products were cloned into lentivirus vector pCDH-CMV-EF1-GFP. The nucleotide sequences of all constructs were verified by DNA sequencing.
Immunohistochemical staining of SALL1 and quantification method
The cell populations of SALL1+ cells in cancer and normal tissues (frozen sections) were determined using immunohistochemical staining with the Histostain®-Plus 3rd Gen IHC Detection Kit (Invitrogen, CA), as we described previously . Immunohistochemical reactions were performed using either mouse monoclonal or rabbit polyclonal antibodies against SALL1 at dilution of 1:1500. Controls were performed by incubating slides with the isotype control antibody instead of primary antibodies, or second antibody alone. SALL1+ cells in tissues were evaluated manually using a computerized image system composed of a Leica ICC50 camera system equipped on a Leica DM750 microscope (North Central Instruments, Minneapolis, MN). Photographs were obtained from 20 randomly selected areas within the tumor tissues of 10 cancer nest areas and 10 cancer stroma areas at a high-power magnification (400 ×). Both cancer nest and stroma areas were counted and summed, and the means of positive cell numbers per field reported.
Reverse-transcription PCR analysis
Total RNA was extracted from tumor or normal tissues and cell lines using Trizol reagent (Invitrogen), and cDNA was transcribed using a SuperScript II RT kit (Invitrogen), both according to manufacturers’ instructions. mRNA expressions of each gene were determined by reverse-transcription PCR using specific primers, and mRNA levels in each samples were normalized to the relative quantity of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All samples were run in triplicate. The primers for each gene used were as following:
SALL1: 5’ TGATGTAGCCAGCATGT 3′ and 5’ AAAGAATTCAGCGCAGCAC 3’.
SALL2: 5’ CCAAGAGTAAAGCGGATGAGA 3′ and 5’ AGTAAGCAGTGCCCAACTCG 3’.
SALL3: 5’ TGGGCCTTCGCTTACTAAAG 3′ and ACAGCAGTGGCAGCTGAAG 3’.
SALL4: 5’ AGCAGCCTCAGCAGCTACC 3′ and 5’ AAGAACTCGGCACAGCATTT 3’.
Cyclin A2: 5’ GGATGGTAGTTTTGAGTCACCAC 3′ and 5’ CACGAGGATAGCTCTCATACTGT 3’.
Cyclin B1: 5’ AACTTTCGCCTGAGCCTATTTT 3′ and 5’ TTGGTCTGACTGCTTGCTCTT 3’.
Cyclin D1: 5’ CAATGACCCCGCACGATTTC 3′ and 5’ CATGGAGGGCGGATTGGAA 3’.
Cyclin E1: 5’ ACCGGTATATGGCGACACAAGAA 3′ and 5’ TCACATACGCAAACTGGTGCAA 3’.
CDK2: 5’ GCATCTTTGCTGAGATGGTGACTC 3′ and 5’ AGTAACTCCTGGCCACACCA 3’.
CDK4: 5’ CATTCTGGTGACAAGTGGTGG 3′ and 5’ TCGGCTTCAGAGTTTCCACAG 3’.
CDK6: 5’ CCAGATGGCTCTAACCTCAGT 3′ and 5 ‘AACTTCCACGAAAAAGAGGCTT 3′.
Cell growth and functional proliferation assay
Tumor cell lines were plated at 2 × 104/well in 24 wells and transfected with one of the following plasmids: pcDNA3.1-SALL1, pcDNA3.1-SALL4, and pcDNA3.1. Cell growth was evaluated at different time points by counting cell numbers. Proliferation assays were performed as previously described . In brief, different numbers of tumor cells (2 × 104, 5 × 104, or 1 × 105) transfected with or without the related genes were cultured in 96-well plates in cell assay medium containing 2% FCS. After 56 h of culture, [3H]-thymidine was added at a final concentration of 1 μCi/well, followed by an additional 16 h of culture. The incorporation of [3H]-thymidine was measured with a liquid scintillation counter.
Cell cycle and apoptosis assays
Transfected cells were cultured for 72 h and apoptosis was analyzed after staining with PE-labeled Annexin V and 7-AAD (BD Biosciences, San Diego, CA). For cell cycle analysis, transfected cells were fixed with 70% ethanol overnight, washed with PBS and incubated with propidium iodide (10 μg/ml) and RNase A (100 μg/ml). Untransfected cells served as controls. All the stained cells were analyzed on a FACSCalibur (BD Bioscience) and the data were analyzed with FlowJo software (Tree Star, Ashland, OR).
Colony formation assay
Five thousand per well of tumor cells infected with lentivirus carrying shRNA against SALL1 or control scramble shRNA, or transfected with SALL1, were seeded in 6-well plates with 0.4% agar for culture. Cell colonies were stained with crystal violet and counted after 2–3 weeks of culture.
Senescence associated β-galactosidase (SA-β-gal) staining
Senescence associated β-Galactosidase (SA-β-Gal) activity in tumor cells was detected as we previously described [31, 32]. Briefly, tumor cell lines were transfected with or without plasmids and cultured for 3 or 5 days. Cells were washed in PBS (pH 7.2), fixed in 3% formaldehyde, and followed to incubate overnight at 37 °C with freshly prepared SA-β-Gal staining solution (1 mg/ml X-gal, 5 mM K3Fe[CN]6, 5 mM K4Fe[CN]6, 2 mM MgCl2 in PBS at pH 6.0). The stained cells were washed with H2O and examined with a microscope. For some experiments, SA-β-Gal+ populations were determined in the transfected tumor cells after exposure to various inhibitors or combined transfection with shRNAs: ATM inhibitor KU55933 (20 μM, Tocris Bioscience); mTOR inhibitor Rapmycin (5 μM, Sigma); MAPK inhibitors U0126 (10 μM), SB203580 (10 μM) and SP600125 (10 μM), or PI3 Kinase inhibitor Wortminnin (10 μM) (Calbiochemistry), or transfection with shRNAs against p38, ERK and mTOR, for 3 or 5 days. The treated tumor cells were then detected for SA-β-Gal expression.
Western-blotting analysis and protein interaction assays
Breast cancer cells transfected with or without plasmids pcDNA3.1-SALL1 or pcDNA3.1-mSALL1, were cultured for 0, 24 h, 48 h and 72 h. Whole cell lysates were prepared for western blotting. The antibodies used in western blotting are as follows: anti-SALL1, anti-ERK, anti-phospho-ERK, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-phospho-p53 (ser15), anti-mTOR, anti-phospho-mTOR; anti-P70S6K, anti-phospho-P70S6K; anti-4E-BP1, anti-phospho-4E-BP1, anti-PTEN, anti-phospho-PTEN and anti-GAPDH rabbit polyclonal antibodies (Cell Signaling Technology, Danvers, MA).
Protein interaction analysis of NuRD complex members with SALL1 (2–137) was performed as previously described [21, 22]. In brief, MCF-7 breast tumor cells were transfected with plasmids pEBG-SALL1, pEBG-SALL1-S2A, and pEBG-SALL1-S2E, and allowed to expression GST-SALL1 fusion proteins for 48–72 h. Cells were incubated for 1 h on ice in lysis buffer (1% Triton X-100, 200 mM sucrose, 50 mM Tris pH 7.4, plus protease cokatail) and the cell suspension was disrupted by sonication. GST-SALL1 fusions and associated protein complexes were isolated by precipitation of 50 μg of total protein with glutathione-Sepahrose beads (Amersham Biosciences) for 2 h at 4 °C. Protein pulldowns were separated by SDS-PAGE and proteins detected by western blot. Primary antibodies were all used at 1:1000 dilution and included: rabbit anti-SALL1, rabbit anti-Mta2 (Abcam, ab 8106), mouse anti-RbAp48 (GeneTex, GTX 70237), rabbit anti-Hdac1 (Abcam, ab 19,845), rabbit anti-Mbd3 (Abcam, ab 157,464). Secondary antibodies were used at 1:10,000 included: goat anti-rabbit (Sigma, A0545) and rabbit anti-mouse (Jackson immune Research 315–035-048).
Flow cytometry analysis
The expression of DNA damage response markers on tumor cells were determined by FACS analysis after staining with anti-human specific antibodies conjugated with either PE or FITC. These human antibodies included: anti-phosphorylated H2Ax, anti-phosphorylated p53bp, and anti-phosphorylated ATM, which were purchased from Cell Signaling Technology or BD Biosciences. All stained cells were analyzed on a FACSCalibur flow cytometer (BD Bioscience) and data analyzed with FlowJo software (Tree Star).
Cell migration and wound healing assay
Breast cancer E0771 and MCF-7 tumor cells transfected with Lenti-SALL1, Lenti-mSALL1 or vector, were plated in 6-well plates and grown to confluence. A wound area was generated by scraping cells with a 200 μl pipette tip across the entire diameter of the dish and extensively rinsed with the medium to remove all cellular debris. Low-serum RPMI 1640 with mitomycin (2 μg/ml) was then added to inhibit cell proliferation during the experiment and the closing of the wound was observed at different time points.
Lentivirus-shRNA generation and gene knockdown in tumor cells
The methods for design and construction of shRNA specific for ERK1, ERK2, P38α, JNK1 and mTOR or scrambled lenti-shRNAs, and generation of recombinant lentivirus carrying GFP and shRNA, have been described previously . shRNAs specific for ATM (TRCN0000194861, TRCN0000039951 and TRCN0000360327), mouse SALL1 (TRCN0000238153, TRCN0000238154 and TRCN0000238155) and human SALL1 (TRCN0000003956, TRCN0000003957 and TRCN0000003958), were purchased from Sigma Aldrich. For lentivirus infection, concentrated lentiviral supernatant with a multiplicity of infection (MOI) of 5–10 in a total volume of 0.5 ml culture medium was added to the tumor cells growing in 24 well plates containing 8 μg/ml polybrene (Sigma), and then centrifuged at 1000 x g for 1 h at room temperature. The infected tumor cells were then transfected with or without pcDNA3.1-SALL-1, and induction of senescence was determined.
In vivo tumorigenesis and metastasis studies
NOD-scid IL2Rγnull (NSG, 6–8 weeks) immunodeficient mice were purchased from The Jackson Laboratory and maintained in the institutional animal facility. All animal studies have been approved by the Institutional Animal Care Committee. For tumorigenesis studies, mouse E0771 (2 × 105/mouse) and B16F0 (1 × 105/mouse) tumor cells infected with lentivirus carrying SALL1, mSALL-1 or vector, were subcutaneously injected into NSG mice. Five mice were included in each group. Tumor size was measured with calipers every 2–3 days. Tumor volume was calculated on the basis of two-dimensional measurements. At the end of experiments, the mice were sacrificed and tumors were isolated and weighted. Furthermore, tumor tissues were embedded into OCT and prepared for cryostat sections (4~ 8 μm), and SA-β-Gal expression was assayed, as described above.
For tumor metastasis studies, lentivirus-transfected E0771 tumor cells were incubated with 100 μg/ml of VivoTag®680 XL (PerkinElmer) for 30 min. Stained tumor cells were washed and then injected intravenously into the tail vein (5 × 104/mouse in 200 μl of buffered saline) into NSG mice. Five mice were included in each group. Mice were imaged with an In Vivo Spectrum Imaging System (IVIS) (Caliper Life Science) at 120 min, and 1, 3, 5, 7, 10, 14, 17 and 19 days post injection. The appropriate filter set for VivoTag®680 XL imaging of 665 nm excitation and 688 nm emission was used. Mice were imaged in the dorsal, right lateral and ventral positions at all the time points. Livers and lungs were harvested at 19 days post injection and stained with 15% black India ink. Visible lung and liver surface macro-metastatic appeared as white spots and were counted using a dissecting microscope. Lungs were collected and fixed in 10% formalin. For tissue morphology and metastasis evaluation, liver and lung tissues were embedded into OCT and frozen sections (4~ 8 μm) were prepared and stained with hematoxylin and eosin (H & E).
Statistical analysis was performed with GraphPad Prism5 software. Unless indicated otherwise, data are expressed as mean ± standard deviation (SD). For public database analysis, The Cancer Genome Atlas (TCGA) normalized log2 transformed breast cancer Argilent microarray expression data sets and methylation database were downloaded from the cBioPortal (http://www.cbioportal.org/)  and used to compare SALL1 mRNA expression among the different breast cancer subtypes and solid normal tissues, as well as analyze methylation difference of SALL1 promoter in COSMIC between the primary tumor in breast cancer and solid normal tissues. The Mann-Whitney U test was utilized for statistical analysis of association between SALL1 expression and breast cancer subtypes. P valve < 0.05 was considered statistically significant. For multiple group comparison in vivo studies, the one-way analysis of variance (ANOVA) was used, followed by the Dunnett’s test for comparing experimental groups against a single control. For single comparison between two groups, paired Student’s t test was used. Nonparametric t-test was chosen if the sample size was too small and did not fit a Gaussian distribution.
The authors would like to thank Dr. Karoly Toth for providing help with mouse imaging studies, and Joy Eslick and Sherri Koehm for FACS sorting and analyses. This work was partially supported by grants from the American Cancer Society (RSG-10-160-01-LIB, to G. P), the Melanoma Research Alliance (to G.P), and the National Institutes of Health (to G. P).
CM, MR and GP: designed research, analyzed data, and wrote the paper. CM, FW, BH, XZ, SF, YJ, LR and SK: performed experiments and prepared figs. EH, YZ, PH, and MV: advised the design of research, collected the tumor samples and clinical information. EH and MV: reviewed the paper. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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