p55PIK Transcriptionally Activated by MZF1 Promotes Colorectal Cancer Cell Proliferation

p55PIK, regulatory subunit of class IA phosphatidylinositol 3-kinase (PI3K), plays a crucial role in cell cycle progression by interaction with tumor repressor retinoblastoma (Rb) protein. A recent study showed that Rb protein can localize to the mitochondria in proliferative cells. Aberrant p55PIK expression may contribute to mitochondrial dysfunction in cancer progression. To reveal the mechanisms of p55PIK transcriptional regulation, the p55PIK promoter characteristics were analyzed. The data show that myeloid zinc finger 1, MZF1, is necessary for p55PIK gene transcription activation. ChIP (Chromatin immuno-precipitation) assay shows that MZF1 binds to the cis-element “TGGGGA” in p55PIK promoter. In MZF1 overexpressed cells, the promoter activity, expression of p55PIK, and cell proliferation rate were observed to be significantly enhanced. Whereas in MZF1-silenced cells, the promoter activity and expression of p55PIK and cell proliferation level was statistically decreased. In CRC tissues, MZF1 and p55PIK mRNA expression were increased (P = 0.046, P = 0.047, resp.). A strong positive correlation (Rs = 0.94) between MZF1 and p55PIK mRNA expression was observed. Taken together, we concluded that p55PIK is transcriptionally activated by MZF1, resulting in increased proliferation of colorectal cancer cells.


Introduction
Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway is thought to play a crucial role in the development of a variety of human cancers. Several academic efforts are underway to de�ne therapeutic inhibitors of the pathway components [1,2]. PI3K interacts with phosphatidylinositol-3-phosphate at the cell membrane and catalyzes the phosphorylation of downstream effector(s) such as Akt [1]. Class IA PI3Ks, consisting of a catalytic subunits p110 and regulatory subunit (p85, p55, and p50), play a critical role in cell proliferation and cell survival [3][4][5][6].
e p55PIK, also known as p55 , is encoded by the pik3r3 gene [7,8]. We previously reported that p55PIK, the N-terminal 24-amino-acid of which is associated with tumor suppressor retinoblastoma protein (Rb), may play an important role in cell cycle control [9]. Ectopic expression of N-terminal 24-amino-acid of p55PIK inhibited cell cycle progression in several cell lines, such as colorectal (HT29) and thyroid (FTC236) cancer cells [10]. One study reported that elevated p55PIK mRNA expression was observed in ovarian, liver, prostate, and breast cancers. A recent study showed that Rb protein can localize to the mitochondria in proliferative cells [11]. Aberrant p55PIK expression may contributes to mitochondrial dysfunction in cancer progression. In addition, apoptosis was observed in p55PIK downregulated ovarian cancer cell lines [12]. Further more, the insulin-like growth factor 2 (IGF2)-p55PIK interaction involved in promoting the growth of a subset of proliferative glioblastomas that lack EGF receptor ampli�cation [13]. ese �ndings suggest that p55PIK was aberrantly expressed in several human cancers and p55PIK may act as an important target for cancer treatment. e detailed mechanism, especially its transcriptional regulation mechanism, remains unknown. Disclosure of the factor(s) that contribute to the regulation of p55PIK expression may be useful in cancer treatment targeting p55PIK. Primer name Sequence pGL3-p55PIK-(−651/+45) up 5 ′ -TTAATTGGTACCGGTCGGGTTGGTTCTTAC-3 ′ pGL3-p55PIK-(−839/+45) up 5 ′ -TTAATTGGTACCGAATCCCGGTACATCG-3 ′ pGL3-p55PIK-(−1064/+45) up 5 ′ -TTAATTGGTACCCACCCACCTTGCCTCT-3 ′ pGL3-p55PIK-(−1243/+45) up Several factor(s) have been reported to regulate p55PIK expression in different human disease models, such as mammary cancer [14] and cerebral ischemia-reperfusion [15]. One study has shown that p55PIK expression increased in the presence of doxorubicin, an anthracycline antibiotic that is used abroad in cancer chemotherapy, in breast cancer MDA-MB-231 cells but not in MCF-7 cells [14]. e genetic factors altered in MDA-MB-231 cells, which are p53/estrogen receptor/progesterone receptor negative, may be involved in regulation of p55PIK expression. Another study reported that the Insulin-like growth factor 2 (IGF2) and p55PIK are overexpressed in more proliferative glioblastomas [13]. In fatty acid and cholesterol biosynthesis, Sterol-regulatory element binding protein-1 (REBP-1) and Platelet-derived growth factor (PDGF) induce the expression of p55PIK in AG01518 human foreskin �broblasts [16]. A recent study revealed that berberine, an effective candidate neuroprotective agent in clinical ischemic stroke, enhances p55PIK promoter activity during cerebral ischemia-reperfusion [15]. In Mycobacterium tuberculosis model of WI-38 cells, downregulated p55PIK expression was observed by recombinant Mycobacterium tuberculosis CFP-10/ESAT-6 protein treatment [17]. Despite the clari�cation of these factors, little is known about the mechanism of p55PIK transcriptional regulation.
e aim of the present study is to identify the ciselements and transcription factor(s) involved in p55PIK transcriptional activation in colorectal cancer cells (CRCs). Firstly, we made in silico analysis and deletion analysis of the p55PIK gene promoter and determined the transcriptional factor(s) that may regulate p55PIK transcription. We also evaluated the in�uence of the transcriptional factors(s) on PI3K expression and the cell growth of CRC cells. Based on the results of this study, the transcription factor(s)-p55PIK axis may be suggested as the potentially crucial target(s) of CRC treatment.

Ethics Statement.
All research involving human participants has been approved by the Huazhong University of Science and Technology Ethics committee. We obtained informed, written consent from all participants involved in this study.

Cell Culture and Transfection.
Cell lines HepG2, HeLa, SW480, and LoVo were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). ese cell lines were cultured at 37 ∘ C in 5% CO 2 /air atmosphere. Transfection was done using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions.

Small
Interfering RNA. Synthetic siRNA targeting human MZF1 (RuiBo, Guangzhou, China) was transfected into cultured cells. Transfection was done using Lipofectamine 2000 following manufacturer's instructions. Cells were cultured in 24-well plates in antibiotic-free 10% fetal bovine serum plus medium and transfected with 50 nmol/L siRNA at 70-80% con�uency. Expression of MZF1 or p55PIK was detected at 24 h or 48 h aer-transfection.

Site-Directed Mutagenesis.
Constructs bearing mutant promoter variants of p55PIK were generated by PCR using the wildtype p55PIK reporter construct (−1243/+45)-p55PIK as template. Underlined nucleotides in Table 2 indicate mutated sequences. Primers were designed according to manufacturer's instructions and produced by Invitrogen. Site-directed mutagenesis was done according to manufacturer's protocol for the Quick Change site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). All mutants of (−1243/+45)-p55PIK were veri�ed correctly by sequencing.

Name
Sequence change Sense primer sequence (5 Chromatin Immunoprecipitation (ChIP) Assay. e chromatin immunoprecipitation (ChIP) assay was used to show interactions between transcription factor(s) and p55PIK promoter DNA sequence in CRC cell lines or tissues samples. In brief, chromatin from SW480 cells or CRC tissues was sheared to DNA fragments with an average size of 200-500 bp. Aer cross-linking reversal and proteinase-K digestion, each individual IP was puri�ed using �IAquick PCR puri�cation kit (�iagen, Valencia, CA, USA) followed by elution with 50 L of elution buffer. Aer elution, IPs were ampli�ed using PCR. To detect the transcription factor(s) binding motif in the p55PIK promoter, we used sense primer 5 ′ -GAAGCCTAGAGAGCGGT-3 ′ and antisense primer 5 ′ -TGTCAAGTGCCTGAGAAC-3 ′ . MZF1 antibody (Santa Cruz, CA, USA) and control IgG (Santa Cruz, CA, USA) were used to immunoprecipitate the protein-DNA complex.

Results and Discussion
3.1. Deletion Analysis of the p55PIK Promoter. p55PIK, the regulatory subunit of class IA phosphatidylinositol 3-kinase (PI3K), plays a crucial role in cell cycle progression. We previously reported that p55PIK, the 24-amino-acid N-terminal end (N24) of which is associated with tumor suppressor retinoblastoma protein (Rb), may play an important role in cell cycle control [9]. A recent study showed that Rb protein can localize to the mitochondria in proliferative cells [11]. Aberrant p55PIK expression may contributes to mitochondrial dysfunction in cancer progression. To reveal the mechanisms of p55PIK transcriptional regulation, the p55PIK promoter characteristics were analyzed.
To identify cis-acting elements within the p55PIK promoter, we constructed a series of luciferase reporter plasmids that contained 5 ′ -deletions of the p55PIK promoter at nucleotides (nt) −1633, −1243, −1064, −839, and −651, with a common 3 ′ -terminus at +45 (Figures 1(a), 1(b)). e promoter activities of the 5 ′ -deletions mutants were assessed by  (Figures 1(c), 1(d)). Importantly, most remarkable changes of promoter activity were observed between constructs (−1243/+45)-p55PIK and (−839/+45)-p55PIK. ese results suggested that p55PIK promoter activity is largely lost upon deletion of the sequence between −1243 and −839 of the full-length promoter. e �rst set of experiments was designed to analy�e the characteristics of p55PIK promoter, and to identify the most commonly activated promoter region of p55PIK in cancer cells, we performed luciferase-based reporter assays. e data demonstrate that the full length of p55PIK promoter located at −1243/+45 upstream of the translation start site (ATG) BioMed Research International   and the −1243/−840 region were responsible for p55PIK gene transcription. We mapped the p55PIK promoter to �nd its most activated fragments. A series of 5 ′ �anking of p55PIK promoter DNA fragments (−1633/+45, −1243/+45, −1064/+45, −839/+45, and −651/+45) were cloned into luciferase-reporter construct and the relative luciferase activity was measured in cancer cells. e results show that −1243/+45 fragment confers highest promoter activity and −840/+45 fragment presents a signi�cant decrease compared with −1243/+45 fragment. e current study indicate that the cis-elements were located at the −1243/−840 region of p55PIK promoter.

3.�. Identi�cation of Cis�Acting Elements Controlling p55PIK
Expression. To identify the critical cis-enhancing elements in the −1243/−840 region, we generated various mutant reporter based on the (−1243/+45)-p55PIK plasmid with substitution mutations by site-directed mutagenesis. As shown in Figure 2(a), the potential transcription factor binding sites of YY1, MZF1, Runx1, ADR1, IRF1, Delta1, and p300 were found in the −1243/−840 region based on motif analysis. e introduction of a TGGGGA site mutation (from TGGGGA to CTAGTG) which located at −901/−896 markedly reduced the luciferase activity of (−1243/+45)-p55PIK (Figure 2(d)), whereas mutation of YY1, RUNX1, or other MZF1 binding sites did not affect the promoter activity of (−1243/+45)-p55PIK (Figures 2(b), 2(c)). ese results demonstrated that the putative MZF1 binding site TGGGGA located at −901/−896 region was crucial for functioning of the p55PIK promoter. e focus of the second set of experiments was to identify the cis-element(s) of p55PIK promoter and corresponding transcription factor(s). e preliminary sequence analysis of the domain −1243/−840 reveals the presence of several nonoverlapping cis-elements and corresponding transcription factors. It was reported that MZF1 binds to the 5 ′ -AGTGGGGA-3 ′ or 5 ′ -CGGGnGAGGGGGAA-3 ′ sequence of gene promoters to regulate the expression of a target gene [19]. YY1, which may bind to the 5 ′ -ACCATTC-3 ′ site of the p55PIK promoter, is a ubiquitously distributed zinc-�nger-type transcription factor, involved in regulating a variety of promoters [16][17][18]. Runx1, which may bind to the 5 ′ -CACCACCC-3 ′ sequence of the p55PIK promoter, is essential for hematopoietic development [19,20]. Interferon regulatory factor 1 (IRF1), which may interact with the 5 ′ -AGACGC-3 ′ DNA binding site, was initially described as a transcription factor able to activate expression of the cytokine interferon beta. IRF-1 plays important roles in immune response [21,22], apoptosis [23,24], and tumor suppression [25]. e p300/CBP coactivator family interacts with transcription factors p53 [26] and STAT3 [27] to transcriptionally activate the expression of their target genes. Only the mutant MZF1-mut1 shows decreased luciferase activity compared with the wildtype promoter plasmids.

MZF1 Binding on the TGGGGA Site Located at −901/−896
Region of the p55PIK Promoter in Colon Cancer Cell and Tissues. Next, to further verify whether MZF1 is involved in p55PIK transcription, we employed primers spanning the putative MZF1-binding site of the p55PIK promoter to perform chromatin immunoprecipitation (ChIP) assays, con�rming the presence of endogenous MZF1 bound to this region in CRC cell line SW480 or CRC tissues. In Figure  2(e), the speci�c sequence within p55PIK promoter was precipitated from cell lysates by anti-MZF1 antibody but not by control IgG in both CRC cell line SW480 and CRC tissues. us, the data strongly indicate that MZF1 binds to TGGGGA domain in the proximal promoter of p55PIK in CRC cells or tissues.

p55PIK Is Transcriptionally Activated by MZF1.
To assess the role of MZF1 in transcriptional activity of p55PIK, we measured the luciferase activity of the p55PIK promoter construct aer transfection of MZF1 expression plasmids or MZF1-SiRNA in CRC cell SW480 and LoVo. Increased luciferase activity was observed in MZF1 overexpressed cells (Figure 3(a)). Figure 3(b) shows decreased luciferase activity aer MZF1 SiRNA transfection. p55PIK mRNA expression was evaluated in MZF1 over-expressing or MZF1-silencing CRC cells. As shown in Figures 3(c) and 3(d), p55PIK mRNA increased aer MZF1 was overexpressed and decreased aer MZF1 was silenced, respectively. By Western Blot analysis, MZF1 over-expressing CRC cell SW480 and LoVo showed increased p55PIK expression (Figures 3(e), 3(f)). Altogether, these �ndings indicate that MZF1 functions at least in part as a transcriptional regulator of p55PIK.

Transcriptional Activation of p55PIK by MZF1 Resulting in Accelerated Cell Proliferation in CRC Cell
Lines. Myeloid �inc �nger 1 (MZF1), a transcription factor belonging to the Kr�ppel �inc �nger protein family, was previously reported as an important factor whose aberrant expression disturbed hematopoietic cell proliferation and cell tumorigenesis [19,28,29]. Transcription factor MZF1, binding on the DNA-binding consensus sequence of 5 ′ -AGTGGGGA-3 ′ or 5 ′ -CGGGnGAGGGGGAA-3 ′ [19], regulated several genes' expression which play important role in cancer migration, invasion, or cancer differentiation. Human liver cancer cell line treated with MZF1 antisense oligonucleotide showed repressed protein kinase C expression and inhibited subcutaneous tumor growth in nude mice [30]. MZF1 transcriptionally regulates Axl receptor tyrosine kinase gene in human colon cancer or cervical cancer, which induced migration and metastasis of colon cancer in vitro and in vivo [31]. mRNA expression of MZF1 and A�L, with signi�cant correlation, were both upregulated in colorectal cancer [31]. Raised cell cycling and loss of contact inhibition were detected in MZF-1 overexpressed NIH 3T3 cells [19]. erefore, MZF1 may function as an oncogene in solid cancer. Next, to determine the effects of the MZF1 induced p55PIK transcriptional activation on the growth, the CRC cell lines were examined with the MTT assay. We found that MZF1-GFP could induce acceleration of the proliferation of CRC (Figure 4(a)) and that MZF1-SiRNA could induce inhibition of growth of CRC (Figure 4(b)) as well.

Relationship between Expression of MZF1 and p55PIK
in CRC Tissues. Finally, to demonstrate the relationship between MZF1 and p55PIK expression in human CRC, we examined endogenous expression of MZF1 and p55PIK in 10 resected CRC tissue samples and corresponding normal mucosal tissues from the same patient received therapeutic surgery. Tumors from 7 of 10 patients showed signi�cant increased expression of MZF1 and p55PIK compared with normal tissues ( and , resp.); MZF1 gene expression was positively and signi�cant correlated with p55PIK expression in the resected tumor tissues (Rs = 0.94; ), indicating that MZF1 and p55PIK are involved in tumorigenesis ( Figure 5).

Conclusion
In summary, we have shown that the transcription factor MZF1, which directly binds to its cis-element within the p55PIK promoter, activates p55PIK expression and acts as a growth accelerator in CRC cells. We also demonstrate that the expression of MZF1 and p55PIK is signi�cant correlated, and they are both overexpressed in resected CRC tissues. is investigation may suggest a strategy for development of therapies on p55PIK-associated cancer especially mitochondrion associated cancer.