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XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity Hong Ji a, 1, Can Huang a, 1, Shourong Wu a, b, c, Vivi Kasim a, b, c, * a The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China b The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing, 400044, China c State and Local Joint Engineering Laboratory for Vascular Implants, Chongqing, 400044, China
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a b s t r a c t
Article history: Received 29 October 2018 Accepted 18 November 2018 Available online xxx
Endoplasmic reticulum (ER) stress activation could be found in a wide range of human tumors. ER stress induces the splicing of X-box binding protein 1 (XBP1) to form its splicing variant XBP1-s, which in turn activates various ER stress-related genes. XBP1-s is highly expressed in various tumors; however, its role in tumorigenesis is still largely unknown. Herein we showed that XBP1-s suppresses the expression of tumor suppressor TAp73, a member of p53 family with high homology with p53, by directly binds to TAp73 promoter and suppresses its transcriptional activity. We also found that overexpression of TAp73 cancelled the effect of XPB1-s on enhancing colorectal cancer cells proliferation and colony formation potential, indicating that TAp73 is critical for XBP1-s-induced tumorigenesis. Together, our findings not only reveal a novel mechanism of TAp73 aberrant regulation in tumor cells, but also link up tumor cells ER stress with tumor suppressive activity of TAp73. © 2018 Elsevier Inc. All rights reserved.
Keywords: X box binding protein 1 (XBP1) Spliced X box binding protein 1 (XBP1-s) TAp73 p53 family Tumorigenesis
1. Introduction Tumor cells typically endure damages attributable to their microenvironments, such as glucose deprivation, hypoxia and DNA damage during tumor initiation and progression. These conditions induce endoplasmic reticulum (ER) stress [1], which promotes protein folding, and thus increases unfolded proteins due to the overload of the ER capacity, leading to the accumulation of unfolded proteins in the ER lumen. To ensure the fidelity of protein folding and to prevent the accumulation of unfolded proteins, cells have evolved the unfolded protein response (UPR) [2]. Thus, UPR is an adaptive response and a defense mechanism through which tumor cells can survive under their severe microenvironments. X-box binding protein 1 (XBP1) is a critical factor for UPR [3]. XBP1 gene is transcribed to mRNA encoding its unspliced form (XBP1-u) protein, the major form of XBP1 under non-ER stress condition. Upon exposure to ER stress, XBP1-u is spliced into its
* Corresponding author. The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, 174 Shazheng Street, Shapingba, Chongqing, 400044, China. E-mail address: [email protected] (V. Kasim). 1 These authors contributed equally to this work.
spliced form (XBP1-s). These two splicing isoforms posses distinct C-terminal region due to the excision of 26 nucleotides of XBP1-u, causing a codon frameshift which leads to the functional disparity of XBP1-u and XBP1-s [3e5]. XBP1-s is widely expressed in various tumors [6e8], and activates the transcription of ER stress-related gene, such as c-Jun N-terminal kinase (JNK), peroxisome proliferator-activated receptor (PPARa), and HIF-1a. XBP1-s plays an important role in promoting tumorigenesis, as it could regulates tumor cell glucose and lipid metabolism, as well as tumor angiogenesis [9]. These evidences indicate that XBP1-s might be a crucial regulator of tumorigenesis; however, its role has not been fully elucidated yet. p73, together with p53 and p63, is a member of tumor suppressor p53 family that share a high homology in their nucleotides and amino acids sequences, as well as remarkable structural similarity [10]. p73 gene encodes two different types of proteins: fulllength isoform (TAp73) and N-terminal truncated isoform (DNp73), which is produced by transcription from an internal promoter or by aberrant splicing of TAp73 mRNA [11]. Unlike DNp73 which function as an oncogene, TAp73 is known to act as a transcription factor and function as a tumor suppressor [12,13]. Recent studies showed that TAp73 control genomic stability through regulation of the spindle assembly checkpoint, and mice deficient
https://doi.org/10.1016/j.bbrc.2018.11.112 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112
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for TAp73 spontaneously develop tumors as a result of mitotic abnormalities [14]. Furthermore, TAp73 could act as an inhibitor of tumor cell migration and invasion, as its deficiency promotes blood vessel formation through up-regulation of proangiogenic and proinflammatory cytokines [15,16]. Similar with that of p53, TAp73 expression decreases in tumors; however, while p53 mutation and enhanced protein degradation is often found in cancer patients, TAp73 mutation is rarely found, and its protein is more stable than that of p53 [17]. These facts prompted the importance of TAp73 aberrant regulatory mechanism in tumor cells. Herein, we identified XBP1-s as a novel upstream regulator of TAp73. XBP1-s could inhibit the transcriptional activity of TAp73 by directly binding to the 244 to 241 region of its promoter. Concomitantly, TAp73 overexpression cancelled XBP1-s-induced colorectal cancer cell proliferation and colony formation potential. Together, these results not only showed a critical function of XBP1s/TAp73 axis in promoting tumorigenesis, but also provided a new insight on the regulatory mechanism of TAp73. Furthermore, these findings emphasize the potential of targeting XBP1-s for cancer therapy.
2.3. RNA extraction, semiquantitative and quantitative RT-PCR analysis Total RNA was extracted with Trizol (Invitrogen Life Technologies) according to the manufacturer's instruction, then 1 mg of the total RNA was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China). For semiquantitative analysis, PCR was performed using cDNA as template, and the amplicons were electrophoresed with agarose gel. Quantitative RT-PCR analysis was performed to assess mRNA expression levels with SYBR Premix Ex Taq (Takara Bio). b-Actin was used for normalization. The sequences of the primers used are listed in Supplementary Table S1. 2.4. Western blotting Cells were collected and lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail, Roche Applied Science, Mannheim, Germany). Western blotting was performed as described previously [21]. The antibodies used are listed in Supplementary Table S2,. Immunoblotting with anti-b-Actin antibody was conducted to ensure equal protein loading.
2. Materials and methods 2.1. Cell culture Human colorectal cancer cell line HCT116 cells were maintained in McCoy's 5A medium (Gibco, Life Technologies, Grand Island, NY). MCF-7 and MCF-10A cells were maintained in Dulbecco's modified Eagle's medium (Gibco). All cell lines were cultured with 10% fetal bovine serum (Biological Industries, Israel) and 1% penicillinstreptomycin. All cell lines were verified using short tandem repeat (STR) profiling method at Cell Bank, Type Culture Collection, Chinese Academy of Science, and tested for mycoplasma contamination using Mycoplasma Detection Kit-Quick Test (Biotool, Houston, TX) routinely every 6 months. Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. For thapsigargin treatment, 1 106 cells were seeded and cultured for 24 h prior to the addition of thapsigargin (Cayman Chemical, Ann Arbor, MI; final concentration 1 nM). Total mRNA and protein were collected 3 h and 6 h after treatment, respectively. For experiments under hypoxia, cells were incubated in a hypoxia chamber (Anaeropouch Box, Mitsubishi GAS Chemical, Tokyo, Japan) for 24 h before RNA or protein extraction [18].
2.2. Vectors construction Long TAp73 reporter vector containing the 4052 to þ438 fragment of human TAp73 promoter at the upstream of firefly luciferase was provided by Dr Massimo Levrero (University of Rome ‘La Sapienza’, Rome, Italy) [19], while the reporter vector bringing the 804 to þ71 fragment of human TAp73 promoter was constructed as described previously [20]. Reporter vectors bringing specific fragments of TAp73 promoter were cloned by amplifying the indicated fragments. TAp73-luciferase vector with mutated XBP1-s binding site (TAp73mut-luc) was constructed base on the site-specific mutagenesis method [21]. XBP1-u (pcXBP1-u), XBP1-s (pcXBP1-s) and FLAG-tagged XBP1-s (FLAG-XBP1-s) overexpression vectors were constructed as described previously [4]. TAp73 overexpression vector (pcp73) was provided by Dr Peng Jiang (Tsinghua University, China) [22].
2.5. Chromatin immunoprecipitation (ChIP) assay ChIP analysis followed by PCR was performed using the ChIP Assay Kit (Beyotime, Guangzhou, China) as described previously [21]. The sequences of the primers used for PCR were: 50 ACATCCCCTGCCCCTTGGATT-30 and 50 -GCTCTGCCCCGCCTCCTT-3'. 2.6. Dual luciferase reporter assays Cells were co-transfected with indicated overexpression vector, reporter vector bringing firefly luciferase, and Renilla luciferase expression vector pRL-SV40 (Promega, Madison, WI) as an internal control. Luciferase reporter activities were measured using Dual Luciferase Reporter Assay (Promega) 24 h after transfection. 2.7. Cell counting assay and crystal violet staining Cells were transfected with indicated vectors as described above. Twenty-four hours after transfection, cells were re-seeded into 96-well plates (2000 cells/well). Total cell numbers were measured by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) at indicated time points. For crystal violet staining, transfected cells were re-seeded in a 96-well plate (2000 cells/well) and cultured for 3 days. Cells were then fixed with 30% ethanol and stained with crystal violet. 2.8. EdU incorporation assay and colony formation assay Cells were transfected with indicated vectors as described above. Twenty-four hours after transfection, EdU incorporation assay was performed using Cell-Light EdU Apollo488 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturer's instruction. Hoechst was used to stain the nuclei. Images were taken with DMI6000B (Leica, Heidelberg, Germany). Quantification of EdU-positive and Hoechst-positive cells was performed using Microsystems LAS AF-TCS MP5 (Leica), and the results are shown as the ratio of EdU-positive cells to Hoechst-positive cells. For colony formation assay, 500 cells were cultured in a six-well plate for 8 days. Cells were then fixed with 30% ethanol and stained with methylene blue. The colonies were then counted.
Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112
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2.9. Statistical analysis Data were expressed as mean ± SD of triplicates. Student's t-test was performed to compare the statistical significance of the differences between two groups. Statistical significance was defined as P < 0.05, and P < 0.01 was considered highly significant.
3. Results 3.1. XBP1-s negatively regulates TAp73 expression in tumor cells XBP1-s, a crucial factor in ER stress and the unfolded protein response, is activated in various tumor cells. Currently the effect of XBP1-s on p53 family is largely unknown. To investigate the relation between XBP1-s and TAp73, we firstly induced XBP1-s accumulation using thapsigargin, an ER stress activator which promotes XBP1 splicing [3], and examined its effect on the expression level of TAp73 in HCT116 cells. Thapsigargin treatment significantly induced the expression levels of both XBP1-s mRNA and protein (Fig. 1A and B); while grossly suppressed those of TAp73. These results indicated the possibility that XBP1-s might be a negative regulator of TAp73. To further confirm this possibility, we next examined the effect of hypoxia on TAp73 expression. Hypoxia is a physiologically important ER stress common to all solid tumors, and had been known to induce XBP1 splicing [7]. Our results showed that compared to the cells cultured under normoxia, hypoxia grossly induced both the mRNA and protein expression levels of XBP1-s,
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while in contrast, decreased those of TAp73 significantly (Fig. 1C and D). XBP1-s and XBP1-u exerts functional disparity, most plausibly due to their distinct C-terminal regions (Fig. 1E). To elucidate the relation between the decrease of TAp73 and the increase of XBP1-s expression, we next examined the effects of overexpressing XBP1-s and XBP1-u on the expression of TAp73. We found that overexpression of XBP1-s, but not XBP1-u, significantly reduced TAp73 mRNA (Fig. 1F and G) and protein (Fig. 1H and I) expression. Together, our results revealed that ER stress suppresses TAp73 expression level, most plausibly by inducing XBP1-s expression. Similar tendency could also be observed in breast cancer lines MCF7, as cells treated with thapsigargin, exposed to hypoxia or overexpressing XBP1-s, showed a robust decrease in TAp73 expression (Figs. S1AeD), indicating that this regulatory mechanism might be common in tumor cells. Furthermore, experiments using normal human breast epithelial cell line MCF-10A also gave similar results (Figs. S1E and F).
3.2. XBP1-s/p73 axis regulates tumor cells proliferation XBP1-s is an oncogene that promotes tumor cells proliferation. Indeed, cell counting assay revealed that XBP1-s overexpression conspicuously increased the number of HCT116 cells (Fig. 2A). Crystal violet staining results also showed a similar tendency (Fig. 2B). Furthermore, we found that XBP1-s overexpression increased the number of EdU positive cells, which represents the proliferative cells, as well as the number of the colonies formed (Fig. 2C and D).
Fig. 1. XBP1-s negatively regulates TAp73 expression. (A and B) mRNA (A) and protein (B) expression levels of XBP1-s and TAp73 in HCT116 cells treated with thapsigargin, as determined by semiquantitative RT-PCR and western blotting, respectively. Cells treated with dimethyl sulfoxide (DMSO) were used as control. (C and D) mRNA (C) and protein (D) expression levels of XBP1-s and TAp73 in HCT116 cells cultured under hypoxia, as determined by semiquantitative RT-PCR and western blotting, respectively. Cells cultured under normoxia were used as control. (E) Schematic diagram showing the homology and the difference between amino acid sequences of XBP1-u and XBP1-s. (FeI) mRNA (F and G) and protein (H and I) expression levels of TAp73 in HCT116 cells overexpressing XBP1-s or XBP1-u, as determined by quantitative RT-PCR (qPCR) and western blotting, respectively. Cells transfected with pcCon were used as control. Quantitative data were shown relative to control and expressed as mean ± s.e.m. of triplicate. *P < 0.05; NS: not significant; pcCon: pcDNA3.1(þ).
Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112
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Fig. 2. XBP1-s/TAp73 axis regulates tumor cells proliferation. (AeB) Total cell number at the indicated time points (A) and crystal violet staining (B) of HCT116 cells overexpressing XBP1-s. (C) Number of proliferating HCT116 cells overexpressing XBP1-s, as determined by the EdU incorporation assay. Representative images (left) and the ratio of EdU positive cells (right) are shown. Hoechst was used to stain the nuclei. Proliferating cells were calculated as the ratio of EdU-positive and Hoechst positive cells, and are shown as relative to the control. Scale bars: 200 mm. (D) Representative images (left) and quantification results (right) of the numbers of colonies formed by HCT116 cells overexpressing XBP1s. (E) Protein expression levels of XBP1-s and TAp73 in HCT116 cells co-transfected with XBP1-s and TAp73 overexpression vectors, as examined using western blotting. (F and G) Total cell number at indicated time points (F) and crystal violet (G) staining of HCT116 cells overexpressing both XBP1-s and TAp73. (H) Number of proliferating HCT116 cells overexpressing both XBP1-s and TAp73, as determined by the EdU incorporation assay. Representative images (left) and the ratio of EdU positive cells (right) are shown. Scale bars: 200 mm. (I) Representative images (left) and quantification results (right) of numbers of the colonies formed by HCT116 cells overexpressing XBP1-s and TAp73. Cells transfected with pcCon were used as control. Quantitative data were shown relative to control and expressed as mean ± s.e.m. of triplicate. *P < 0.05; **P < 0.01; pcCon: pcDNA3.1(þ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
To investigate the role of TAp73 in XBP1-s-induced tumor cell proliferation and colony formation, we overexpressed both XBP1-s and TAp73 (Fig. 2E). TAp73 overexpression cancelled the increase of cell numbers induced by XBP1-s (Fig. 2F and G), as well as the effect of XBP1-s in promoting HCT116 cells proliferation and colony formation potential (Fig. 2H and I). Taken together, these results clearly showed that TAp73 is critical for XBP1-s-induced tumor cells proliferation and colony formation.
To confirm that XBP1-s regulation on the above-mentioned genes occurs via its negative regulation on TAp73, we examined the expression levels of TAp73 target genes in HCT116 cells overexpressing both XBP1-s and TAp73. We found that TAp73 overexpression restored the expression levels of Bim and Bak suppressed by XBP1-s overexpression, and cancelled the increase of Twist and Snail expression (Fig. 3EeH). These results further confirmed that XBP1-s is a novel negative regulator of TAp73.
3.3. XBP1-s regulates TAp73 target genes
3.4. XBP1-s regulates TAp73 transcription by directly binds to its promoter region
Previous research showed that TAp73 can induce the expression of apoptosis factors Bim and Bak [23], while inhibiting the expression of epithelial-to-mesenchymal transition (EMT) factors Twist and Snail [24]. We then investigated the effect of XBP1-s on these TAp73 target genes, and found that XBP1-s overexpression significantly suppressed the expression levels of Bim and Bak (Fig. 3A and B), while grossly induced the expression levels of Twist and Snail (Fig. 3C and D).
To further reveal the underlying mechanism of XBP1-s regulation on TAp73 transcription, we next examined whether XBP1-s affects TAp73 transcriptional activity. To this end, we used two luciferase reporter assay vectors bringing the 4052 to þ438 (Long TAp73 reporter) or the 804 to þ71 (Short TAp73 reporter) regions of TAp73 promoter (Fig. 4A, left). Dual luciferase reporter assay results clearly showed that overexpression of XBP1-s could
Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112
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Fig. 3. XBP1-s regulates TAp73 downstream factors. (AeD) mRNA expression levels of Bim (A), Bak (B), Twist (C) and Snail (D) in HCT116 cells overexpressing XBP1-s were examined using qPCR. (EeH) mRNA expression levels of Bim (E), Bak (F), Twist (G) and Snail (H) in HCT116 cells overexpressing both XBP1-s and TAp73, as determined by qPCR. Cells transfected with pcCon were used as control. Quantitative data were shown relative to control and expressed as mean ± s.e.m. of triplicate. *P < 0.05; **P < 0.01; pcCon: pcDNA3.1(þ).
suppress the activities of both of two TAp73 reporters to a similar extent (Fig. 4A, right), indicating that XBP1-s regulation on TAp73 expression occurs in its transcriptional level, and that the regulation region is located between 804 and þ71 region of TAp73 promoter. To assess whether this regulation was also common in other cells, we overexpressed XBP1-s in MCF-7 and MCF-10A cells, and found that in XBP1-s overexpression grossly suppressed the activities of TAp73 reporter (Supplementary Figs. S2A and B). Previous study has reported that XBP1-s could bind to GACG sequence in the promoter of its target gene. Indeed, we found such a binding sequence in the 244 to 241 region of TAp73 promoter. To reveal whether XBP1-s could bind directly to the TAp73 promoter, we performed chromatin immunoprecipitation (ChIP) assay followed by PCR using a primer set targeting the 513 to 203 region of the TAp73 promoter. ChIP assay results clearly showed that XBP1-s could bind to the 513 to 203 region of the TAp73 promoter (Fig. 4B). Next, to map the XBP1-s responsive region within the TAp73 promoter, we constructed a series of 50 -deleted TAp73 promoterdriven luciferase reporter vector bringing the 513 to þ71 (TAp73-luc-del-1), 416 to þ71 (TAp73-luc-del-2), 319 to þ71 (TAp73-luc-del-3) and 221 to þ71 (TAp73-luc-del-4) regions of TAp73 promoter (Fig. 4C, left). Our results showed that while XBP1s overexpression still could robustly suppressed the activities of TAp73-luc-del-1, TAp73-luc-del-2 and TAp73-luc-del-3 reporter vectors to a level similar to that of short TAp73 reporter vector (804 to þ71); it failed to significantly affect the activity of TAp73luc-del-4 reporter vector (Fig. 4C, right), indicating that the region between 319 and 222 is critical for XBP1-s inhibition on TAp73 expression. Finally, to assess whether the predicted binding site (244 to 241) is functional, we constructed a TAp73 luciferase reporter
vector with three point mutations in the XBP1-s core binding site (TAp73mut-luc): the GACG sequence in the wild-type TAp73 promoter was mutated into GGTA (Fig. 4D, left). We found that while XBP1-s overexpression could robustly suppress the activity of the wild-type TAp73 reporter vector (short TAp73-luc), this effect was diminished when TAp73mut-luc was used (Fig. 4D, right). These results strongly indicate that XBP1-s could directly bind to the TAp73 promoter and suppresses its transcriptional activity, and that the 244 to 241 region in TAp73 promoter is critical for such regulation. Together, our results clearly showed that XBP1-s is a novel regulator of TAp73 that suppresses its transcriptional activity through a direct binding, and that XBP1-s/TAp73 axis plays an important role in regulating tumor cells proliferation (Fig. 4E). 4. Discussion The physiological environment of solid tumors differs from that of normal tissues. They usually faced severe microenvironment including hypoxia, low pH and lack of nutrients, leading to the activation of ER stress and UPR as an adaptive mechanism to support tumor cells proliferation [25]. Our findings revealed a novel regulatory mechanism of tumor suppressor TAp73 by XBP1-s. XBP1-s directly binds to TAp73 promoter, thereby suppresses its transcriptional activity, and subsequently promotes tumor cells proliferation and colony formation. Indeed, while its downregulation is commonly found in human cancers, TAp73 is rarely mutated [17], emphasizing the importance of elucidating its aberrant regulatory mechanism in tumors. TAp73 is a member of tumor suppressor p53 family, which plays a pivotal role in preventing the development of tumor by inducing apoptosis, DNA repair and cell-cycle arrest [26]. Although TAp73
Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112
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Fig. 4. XBP1-s affects TAp73 transcriptional activity by directly bind to its promoter region. (A) Schematic diagram (left) and relative luciferase activities of TAp73 reporter vectors in HCT116 cells overexpressing XBP1-s. (B) Binding of XBP1-s to the promoter region of TAp73 was examined using chromatin immunoprecipitation assay with anti-FLAG antibody followed by PCR. The locations of primer set used for PCR were shown. (C) Schematic diagram (left) and relative luciferase activities of serial TAp73 promoter-driven luciferase reporters (right) in XBP1-s-overexpressed HCT116 cells. (D) Schematic diagram (left) and luciferase activities of Short TAp73-luc and TAp73mut-luc reporter vectors in XBP1-s-overexpressed HCT116 cells. Mutated base pairs were indicated in red. Cells transfected with pcCon were used as control. (E) Schematic diagram showing the mechanism of XBP1-s regulation on TAp73. Luciferase activities were measured using dual luciferase assay, shown relative to control, and expressed as mean ± s.e.m. of triplicate. **P < 0.01; NS: not significant; pcCon: pcDNA3.1(þ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
shares many target genes with p53 in exerting its tumor suppressive functions, previous reports had shown their distinct regulatory mechanisms. Dulloo et al. found that mouse double minute 2 (MDM2) promotes the ubiquitination and the proteasomal degradation of p53; however, it inhibits the activity of TAp73 as a transcriptonal factor [27]. Furthermore, we previously found that while XBP1-u could binds and stabilizes MDM2 protein by suppressing its self-ubiquitination, and thereby enhances p53 degradation, XBP1-s does not significantly regulate p53 [4]. In contrast, our current results showed that while XBP1-u could not significantly regulate TAp73 expression, XBP1-s remarkably suppresses TAp73
transcription by directly binds to its promoter region. Thus, the two splicing variants of XBP1, XBP1-u and XBP1-s, exert completely different and specific mechanisms in regulating p53 family members: XBP1-u regulates p53 post-translationally by destabilizing its protein, whereas XBP1-s suppresses TAp73 transcriptional activity. The functional differences of the two isoforms of XBP1 are most plausibly due to their distinct C-terminal, as 26 bp of nucleotides in XBP1-u are excised during splicing, causing a codon shift in XBP1-s [4,5]. Together with the fact that the levels of both XBP1-u and XBP1-s increase in tumor tissues [4,8], our results strongly suggest that both of them are crucial for tumorigenesis by exerting their
Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112
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specific functions in suppressing p53 and TAp73. In conclusion, in this study we unraveled a novel role of XBP1-s in regulating TAp73, as well as the importance of XBP1-s/TAp73 axis in promoting colorectal cancer cell proliferation and colony formation. Our findings not only provide novel insights regarding the aberrant TAp73 regulation in tumors, but also link up tumor cells ER stress with the tumor suppressive activity of TAp73. Moreover, these findings also suggest the potential of targeting XBP1-s/TAp73 axis for treating cancers.
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Conflicts of interest
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The authors declare no conflict of interest. Acknowledgements This work was supported by the grants from the Natural Science Foundation of Chongqing No. cstc2018jcyjAX0411 and cstc2018jcyjAX0374, and the Graduate Scientific Research and Innovation Foundation of Chongqing (CYB17039). We thank Dr Peng Jiang (Tsinghua University, China) for providing TAp73 overexpression vector; and Dr Massimo Levrero (University of Rome ‘La Sapienza’, Rome, Italy) for providing long TAp73 reporter.
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Appendix A. Supplementary data [18]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.112. Transparency document
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.112.
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XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity Hong Ji a, 1, Can Huang a, 1, Shourong Wu a, b, c, Vivi Kasim a, b, c, * a The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China b The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing, 400044, China c State and Local Joint Engineering Laboratory for Vascular Implants, Chongqing, 400044, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 October 2018 Accepted 18 November 2018 Available online xxx
Endoplasmic reticulum (ER) stress activation could be found in a wide range of human tumors. ER stress induces the splicing of X-box binding protein 1 (XBP1) to form its splicing variant XBP1-s, which in turn activates various ER stress-related genes. XBP1-s is highly expressed in various tumors; however, its role in tumorigenesis is still largely unknown. Herein we showed that XBP1-s suppresses the expression of tumor suppressor TAp73, a member of p53 family with high homology with p53, by directly binds to TAp73 promoter and suppresses its transcriptional activity. We also found that overexpression of TAp73 cancelled the effect of XPB1-s on enhancing colorectal cancer cells proliferation and colony formation potential, indicating that TAp73 is critical for XBP1-s-induced tumorigenesis. Together, our findings not only reveal a novel mechanism of TAp73 aberrant regulation in tumor cells, but also link up tumor cells ER stress with tumor suppressive activity of TAp73. © 2018 Elsevier Inc. All rights reserved.
Keywords: X box binding protein 1 (XBP1) Spliced X box binding protein 1 (XBP1-s) TAp73 p53 family Tumorigenesis
1. Introduction Tumor cells typically endure damages attributable to their microenvironments, such as glucose deprivation, hypoxia and DNA damage during tumor initiation and progression. These conditions induce endoplasmic reticulum (ER) stress [1], which promotes protein folding, and thus increases unfolded proteins due to the overload of the ER capacity, leading to the accumulation of unfolded proteins in the ER lumen. To ensure the fidelity of protein folding and to prevent the accumulation of unfolded proteins, cells have evolved the unfolded protein response (UPR) [2]. Thus, UPR is an adaptive response and a defense mechanism through which tumor cells can survive under their severe microenvironments. X-box binding protein 1 (XBP1) is a critical factor for UPR [3]. XBP1 gene is transcribed to mRNA encoding its unspliced form (XBP1-u) protein, the major form of XBP1 under non-ER stress condition. Upon exposure to ER stress, XBP1-u is spliced into its
* Corresponding author. The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, 174 Shazheng Street, Shapingba, Chongqing, 400044, China. E-mail address: [email protected] (V. Kasim). 1 These authors contributed equally to this work.
spliced form (XBP1-s). These two splicing isoforms posses distinct C-terminal region due to the excision of 26 nucleotides of XBP1-u, causing a codon frameshift which leads to the functional disparity of XBP1-u and XBP1-s [3e5]. XBP1-s is widely expressed in various tumors [6e8], and activates the transcription of ER stress-related gene, such as c-Jun N-terminal kinase (JNK), peroxisome proliferator-activated receptor (PPARa), and HIF-1a. XBP1-s plays an important role in promoting tumorigenesis, as it could regulates tumor cell glucose and lipid metabolism, as well as tumor angiogenesis [9]. These evidences indicate that XBP1-s might be a crucial regulator of tumorigenesis; however, its role has not been fully elucidated yet. p73, together with p53 and p63, is a member of tumor suppressor p53 family that share a high homology in their nucleotides and amino acids sequences, as well as remarkable structural similarity [10]. p73 gene encodes two different types of proteins: fulllength isoform (TAp73) and N-terminal truncated isoform (DNp73), which is produced by transcription from an internal promoter or by aberrant splicing of TAp73 mRNA [11]. Unlike DNp73 which function as an oncogene, TAp73 is known to act as a transcription factor and function as a tumor suppressor [12,13]. Recent studies showed that TAp73 control genomic stability through regulation of the spindle assembly checkpoint, and mice deficient
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Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112
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for TAp73 spontaneously develop tumors as a result of mitotic abnormalities [14]. Furthermore, TAp73 could act as an inhibitor of tumor cell migration and invasion, as its deficiency promotes blood vessel formation through up-regulation of proangiogenic and proinflammatory cytokines [15,16]. Similar with that of p53, TAp73 expression decreases in tumors; however, while p53 mutation and enhanced protein degradation is often found in cancer patients, TAp73 mutation is rarely found, and its protein is more stable than that of p53 [17]. These facts prompted the importance of TAp73 aberrant regulatory mechanism in tumor cells. Herein, we identified XBP1-s as a novel upstream regulator of TAp73. XBP1-s could inhibit the transcriptional activity of TAp73 by directly binding to the 244 to 241 region of its promoter. Concomitantly, TAp73 overexpression cancelled XBP1-s-induced colorectal cancer cell proliferation and colony formation potential. Together, these results not only showed a critical function of XBP1s/TAp73 axis in promoting tumorigenesis, but also provided a new insight on the regulatory mechanism of TAp73. Furthermore, these findings emphasize the potential of targeting XBP1-s for cancer therapy.
2.3. RNA extraction, semiquantitative and quantitative RT-PCR analysis Total RNA was extracted with Trizol (Invitrogen Life Technologies) according to the manufacturer's instruction, then 1 mg of the total RNA was reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China). For semiquantitative analysis, PCR was performed using cDNA as template, and the amplicons were electrophoresed with agarose gel. Quantitative RT-PCR analysis was performed to assess mRNA expression levels with SYBR Premix Ex Taq (Takara Bio). b-Actin was used for normalization. The sequences of the primers used are listed in Supplementary Table S1. 2.4. Western blotting Cells were collected and lysed with RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktail (complete cocktail, Roche Applied Science, Mannheim, Germany). Western blotting was performed as described previously [21]. The antibodies used are listed in Supplementary Table S2,. Immunoblotting with anti-b-Actin antibody was conducted to ensure equal protein loading.
2. Materials and methods 2.1. Cell culture Human colorectal cancer cell line HCT116 cells were maintained in McCoy's 5A medium (Gibco, Life Technologies, Grand Island, NY). MCF-7 and MCF-10A cells were maintained in Dulbecco's modified Eagle's medium (Gibco). All cell lines were cultured with 10% fetal bovine serum (Biological Industries, Israel) and 1% penicillinstreptomycin. All cell lines were verified using short tandem repeat (STR) profiling method at Cell Bank, Type Culture Collection, Chinese Academy of Science, and tested for mycoplasma contamination using Mycoplasma Detection Kit-Quick Test (Biotool, Houston, TX) routinely every 6 months. Transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. For thapsigargin treatment, 1 106 cells were seeded and cultured for 24 h prior to the addition of thapsigargin (Cayman Chemical, Ann Arbor, MI; final concentration 1 nM). Total mRNA and protein were collected 3 h and 6 h after treatment, respectively. For experiments under hypoxia, cells were incubated in a hypoxia chamber (Anaeropouch Box, Mitsubishi GAS Chemical, Tokyo, Japan) for 24 h before RNA or protein extraction [18].
2.2. Vectors construction Long TAp73 reporter vector containing the 4052 to þ438 fragment of human TAp73 promoter at the upstream of firefly luciferase was provided by Dr Massimo Levrero (University of Rome ‘La Sapienza’, Rome, Italy) [19], while the reporter vector bringing the 804 to þ71 fragment of human TAp73 promoter was constructed as described previously [20]. Reporter vectors bringing specific fragments of TAp73 promoter were cloned by amplifying the indicated fragments. TAp73-luciferase vector with mutated XBP1-s binding site (TAp73mut-luc) was constructed base on the site-specific mutagenesis method [21]. XBP1-u (pcXBP1-u), XBP1-s (pcXBP1-s) and FLAG-tagged XBP1-s (FLAG-XBP1-s) overexpression vectors were constructed as described previously [4]. TAp73 overexpression vector (pcp73) was provided by Dr Peng Jiang (Tsinghua University, China) [22].
2.5. Chromatin immunoprecipitation (ChIP) assay ChIP analysis followed by PCR was performed using the ChIP Assay Kit (Beyotime, Guangzhou, China) as described previously [21]. The sequences of the primers used for PCR were: 50 ACATCCCCTGCCCCTTGGATT-30 and 50 -GCTCTGCCCCGCCTCCTT-3'. 2.6. Dual luciferase reporter assays Cells were co-transfected with indicated overexpression vector, reporter vector bringing firefly luciferase, and Renilla luciferase expression vector pRL-SV40 (Promega, Madison, WI) as an internal control. Luciferase reporter activities were measured using Dual Luciferase Reporter Assay (Promega) 24 h after transfection. 2.7. Cell counting assay and crystal violet staining Cells were transfected with indicated vectors as described above. Twenty-four hours after transfection, cells were re-seeded into 96-well plates (2000 cells/well). Total cell numbers were measured by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) at indicated time points. For crystal violet staining, transfected cells were re-seeded in a 96-well plate (2000 cells/well) and cultured for 3 days. Cells were then fixed with 30% ethanol and stained with crystal violet. 2.8. EdU incorporation assay and colony formation assay Cells were transfected with indicated vectors as described above. Twenty-four hours after transfection, EdU incorporation assay was performed using Cell-Light EdU Apollo488 In Vitro Imaging Kit (RiboBio, Guangzhou, China) according to the manufacturer's instruction. Hoechst was used to stain the nuclei. Images were taken with DMI6000B (Leica, Heidelberg, Germany). Quantification of EdU-positive and Hoechst-positive cells was performed using Microsystems LAS AF-TCS MP5 (Leica), and the results are shown as the ratio of EdU-positive cells to Hoechst-positive cells. For colony formation assay, 500 cells were cultured in a six-well plate for 8 days. Cells were then fixed with 30% ethanol and stained with methylene blue. The colonies were then counted.
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2.9. Statistical analysis Data were expressed as mean ± SD of triplicates. Student's t-test was performed to compare the statistical significance of the differences between two groups. Statistical significance was defined as P < 0.05, and P < 0.01 was considered highly significant.
3. Results 3.1. XBP1-s negatively regulates TAp73 expression in tumor cells XBP1-s, a crucial factor in ER stress and the unfolded protein response, is activated in various tumor cells. Currently the effect of XBP1-s on p53 family is largely unknown. To investigate the relation between XBP1-s and TAp73, we firstly induced XBP1-s accumulation using thapsigargin, an ER stress activator which promotes XBP1 splicing [3], and examined its effect on the expression level of TAp73 in HCT116 cells. Thapsigargin treatment significantly induced the expression levels of both XBP1-s mRNA and protein (Fig. 1A and B); while grossly suppressed those of TAp73. These results indicated the possibility that XBP1-s might be a negative regulator of TAp73. To further confirm this possibility, we next examined the effect of hypoxia on TAp73 expression. Hypoxia is a physiologically important ER stress common to all solid tumors, and had been known to induce XBP1 splicing [7]. Our results showed that compared to the cells cultured under normoxia, hypoxia grossly induced both the mRNA and protein expression levels of XBP1-s,
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while in contrast, decreased those of TAp73 significantly (Fig. 1C and D). XBP1-s and XBP1-u exerts functional disparity, most plausibly due to their distinct C-terminal regions (Fig. 1E). To elucidate the relation between the decrease of TAp73 and the increase of XBP1-s expression, we next examined the effects of overexpressing XBP1-s and XBP1-u on the expression of TAp73. We found that overexpression of XBP1-s, but not XBP1-u, significantly reduced TAp73 mRNA (Fig. 1F and G) and protein (Fig. 1H and I) expression. Together, our results revealed that ER stress suppresses TAp73 expression level, most plausibly by inducing XBP1-s expression. Similar tendency could also be observed in breast cancer lines MCF7, as cells treated with thapsigargin, exposed to hypoxia or overexpressing XBP1-s, showed a robust decrease in TAp73 expression (Figs. S1AeD), indicating that this regulatory mechanism might be common in tumor cells. Furthermore, experiments using normal human breast epithelial cell line MCF-10A also gave similar results (Figs. S1E and F).
3.2. XBP1-s/p73 axis regulates tumor cells proliferation XBP1-s is an oncogene that promotes tumor cells proliferation. Indeed, cell counting assay revealed that XBP1-s overexpression conspicuously increased the number of HCT116 cells (Fig. 2A). Crystal violet staining results also showed a similar tendency (Fig. 2B). Furthermore, we found that XBP1-s overexpression increased the number of EdU positive cells, which represents the proliferative cells, as well as the number of the colonies formed (Fig. 2C and D).
Fig. 1. XBP1-s negatively regulates TAp73 expression. (A and B) mRNA (A) and protein (B) expression levels of XBP1-s and TAp73 in HCT116 cells treated with thapsigargin, as determined by semiquantitative RT-PCR and western blotting, respectively. Cells treated with dimethyl sulfoxide (DMSO) were used as control. (C and D) mRNA (C) and protein (D) expression levels of XBP1-s and TAp73 in HCT116 cells cultured under hypoxia, as determined by semiquantitative RT-PCR and western blotting, respectively. Cells cultured under normoxia were used as control. (E) Schematic diagram showing the homology and the difference between amino acid sequences of XBP1-u and XBP1-s. (FeI) mRNA (F and G) and protein (H and I) expression levels of TAp73 in HCT116 cells overexpressing XBP1-s or XBP1-u, as determined by quantitative RT-PCR (qPCR) and western blotting, respectively. Cells transfected with pcCon were used as control. Quantitative data were shown relative to control and expressed as mean ± s.e.m. of triplicate. *P < 0.05; NS: not significant; pcCon: pcDNA3.1(þ).
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Fig. 2. XBP1-s/TAp73 axis regulates tumor cells proliferation. (AeB) Total cell number at the indicated time points (A) and crystal violet staining (B) of HCT116 cells overexpressing XBP1-s. (C) Number of proliferating HCT116 cells overexpressing XBP1-s, as determined by the EdU incorporation assay. Representative images (left) and the ratio of EdU positive cells (right) are shown. Hoechst was used to stain the nuclei. Proliferating cells were calculated as the ratio of EdU-positive and Hoechst positive cells, and are shown as relative to the control. Scale bars: 200 mm. (D) Representative images (left) and quantification results (right) of the numbers of colonies formed by HCT116 cells overexpressing XBP1s. (E) Protein expression levels of XBP1-s and TAp73 in HCT116 cells co-transfected with XBP1-s and TAp73 overexpression vectors, as examined using western blotting. (F and G) Total cell number at indicated time points (F) and crystal violet (G) staining of HCT116 cells overexpressing both XBP1-s and TAp73. (H) Number of proliferating HCT116 cells overexpressing both XBP1-s and TAp73, as determined by the EdU incorporation assay. Representative images (left) and the ratio of EdU positive cells (right) are shown. Scale bars: 200 mm. (I) Representative images (left) and quantification results (right) of numbers of the colonies formed by HCT116 cells overexpressing XBP1-s and TAp73. Cells transfected with pcCon were used as control. Quantitative data were shown relative to control and expressed as mean ± s.e.m. of triplicate. *P < 0.05; **P < 0.01; pcCon: pcDNA3.1(þ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
To investigate the role of TAp73 in XBP1-s-induced tumor cell proliferation and colony formation, we overexpressed both XBP1-s and TAp73 (Fig. 2E). TAp73 overexpression cancelled the increase of cell numbers induced by XBP1-s (Fig. 2F and G), as well as the effect of XBP1-s in promoting HCT116 cells proliferation and colony formation potential (Fig. 2H and I). Taken together, these results clearly showed that TAp73 is critical for XBP1-s-induced tumor cells proliferation and colony formation.
To confirm that XBP1-s regulation on the above-mentioned genes occurs via its negative regulation on TAp73, we examined the expression levels of TAp73 target genes in HCT116 cells overexpressing both XBP1-s and TAp73. We found that TAp73 overexpression restored the expression levels of Bim and Bak suppressed by XBP1-s overexpression, and cancelled the increase of Twist and Snail expression (Fig. 3EeH). These results further confirmed that XBP1-s is a novel negative regulator of TAp73.
3.3. XBP1-s regulates TAp73 target genes
3.4. XBP1-s regulates TAp73 transcription by directly binds to its promoter region
Previous research showed that TAp73 can induce the expression of apoptosis factors Bim and Bak [23], while inhibiting the expression of epithelial-to-mesenchymal transition (EMT) factors Twist and Snail [24]. We then investigated the effect of XBP1-s on these TAp73 target genes, and found that XBP1-s overexpression significantly suppressed the expression levels of Bim and Bak (Fig. 3A and B), while grossly induced the expression levels of Twist and Snail (Fig. 3C and D).
To further reveal the underlying mechanism of XBP1-s regulation on TAp73 transcription, we next examined whether XBP1-s affects TAp73 transcriptional activity. To this end, we used two luciferase reporter assay vectors bringing the 4052 to þ438 (Long TAp73 reporter) or the 804 to þ71 (Short TAp73 reporter) regions of TAp73 promoter (Fig. 4A, left). Dual luciferase reporter assay results clearly showed that overexpression of XBP1-s could
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Fig. 3. XBP1-s regulates TAp73 downstream factors. (AeD) mRNA expression levels of Bim (A), Bak (B), Twist (C) and Snail (D) in HCT116 cells overexpressing XBP1-s were examined using qPCR. (EeH) mRNA expression levels of Bim (E), Bak (F), Twist (G) and Snail (H) in HCT116 cells overexpressing both XBP1-s and TAp73, as determined by qPCR. Cells transfected with pcCon were used as control. Quantitative data were shown relative to control and expressed as mean ± s.e.m. of triplicate. *P < 0.05; **P < 0.01; pcCon: pcDNA3.1(þ).
suppress the activities of both of two TAp73 reporters to a similar extent (Fig. 4A, right), indicating that XBP1-s regulation on TAp73 expression occurs in its transcriptional level, and that the regulation region is located between 804 and þ71 region of TAp73 promoter. To assess whether this regulation was also common in other cells, we overexpressed XBP1-s in MCF-7 and MCF-10A cells, and found that in XBP1-s overexpression grossly suppressed the activities of TAp73 reporter (Supplementary Figs. S2A and B). Previous study has reported that XBP1-s could bind to GACG sequence in the promoter of its target gene. Indeed, we found such a binding sequence in the 244 to 241 region of TAp73 promoter. To reveal whether XBP1-s could bind directly to the TAp73 promoter, we performed chromatin immunoprecipitation (ChIP) assay followed by PCR using a primer set targeting the 513 to 203 region of the TAp73 promoter. ChIP assay results clearly showed that XBP1-s could bind to the 513 to 203 region of the TAp73 promoter (Fig. 4B). Next, to map the XBP1-s responsive region within the TAp73 promoter, we constructed a series of 50 -deleted TAp73 promoterdriven luciferase reporter vector bringing the 513 to þ71 (TAp73-luc-del-1), 416 to þ71 (TAp73-luc-del-2), 319 to þ71 (TAp73-luc-del-3) and 221 to þ71 (TAp73-luc-del-4) regions of TAp73 promoter (Fig. 4C, left). Our results showed that while XBP1s overexpression still could robustly suppressed the activities of TAp73-luc-del-1, TAp73-luc-del-2 and TAp73-luc-del-3 reporter vectors to a level similar to that of short TAp73 reporter vector (804 to þ71); it failed to significantly affect the activity of TAp73luc-del-4 reporter vector (Fig. 4C, right), indicating that the region between 319 and 222 is critical for XBP1-s inhibition on TAp73 expression. Finally, to assess whether the predicted binding site (244 to 241) is functional, we constructed a TAp73 luciferase reporter
vector with three point mutations in the XBP1-s core binding site (TAp73mut-luc): the GACG sequence in the wild-type TAp73 promoter was mutated into GGTA (Fig. 4D, left). We found that while XBP1-s overexpression could robustly suppress the activity of the wild-type TAp73 reporter vector (short TAp73-luc), this effect was diminished when TAp73mut-luc was used (Fig. 4D, right). These results strongly indicate that XBP1-s could directly bind to the TAp73 promoter and suppresses its transcriptional activity, and that the 244 to 241 region in TAp73 promoter is critical for such regulation. Together, our results clearly showed that XBP1-s is a novel regulator of TAp73 that suppresses its transcriptional activity through a direct binding, and that XBP1-s/TAp73 axis plays an important role in regulating tumor cells proliferation (Fig. 4E). 4. Discussion The physiological environment of solid tumors differs from that of normal tissues. They usually faced severe microenvironment including hypoxia, low pH and lack of nutrients, leading to the activation of ER stress and UPR as an adaptive mechanism to support tumor cells proliferation [25]. Our findings revealed a novel regulatory mechanism of tumor suppressor TAp73 by XBP1-s. XBP1-s directly binds to TAp73 promoter, thereby suppresses its transcriptional activity, and subsequently promotes tumor cells proliferation and colony formation. Indeed, while its downregulation is commonly found in human cancers, TAp73 is rarely mutated [17], emphasizing the importance of elucidating its aberrant regulatory mechanism in tumors. TAp73 is a member of tumor suppressor p53 family, which plays a pivotal role in preventing the development of tumor by inducing apoptosis, DNA repair and cell-cycle arrest [26]. Although TAp73
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Fig. 4. XBP1-s affects TAp73 transcriptional activity by directly bind to its promoter region. (A) Schematic diagram (left) and relative luciferase activities of TAp73 reporter vectors in HCT116 cells overexpressing XBP1-s. (B) Binding of XBP1-s to the promoter region of TAp73 was examined using chromatin immunoprecipitation assay with anti-FLAG antibody followed by PCR. The locations of primer set used for PCR were shown. (C) Schematic diagram (left) and relative luciferase activities of serial TAp73 promoter-driven luciferase reporters (right) in XBP1-s-overexpressed HCT116 cells. (D) Schematic diagram (left) and luciferase activities of Short TAp73-luc and TAp73mut-luc reporter vectors in XBP1-s-overexpressed HCT116 cells. Mutated base pairs were indicated in red. Cells transfected with pcCon were used as control. (E) Schematic diagram showing the mechanism of XBP1-s regulation on TAp73. Luciferase activities were measured using dual luciferase assay, shown relative to control, and expressed as mean ± s.e.m. of triplicate. **P < 0.01; NS: not significant; pcCon: pcDNA3.1(þ). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
shares many target genes with p53 in exerting its tumor suppressive functions, previous reports had shown their distinct regulatory mechanisms. Dulloo et al. found that mouse double minute 2 (MDM2) promotes the ubiquitination and the proteasomal degradation of p53; however, it inhibits the activity of TAp73 as a transcriptonal factor [27]. Furthermore, we previously found that while XBP1-u could binds and stabilizes MDM2 protein by suppressing its self-ubiquitination, and thereby enhances p53 degradation, XBP1-s does not significantly regulate p53 [4]. In contrast, our current results showed that while XBP1-u could not significantly regulate TAp73 expression, XBP1-s remarkably suppresses TAp73
transcription by directly binds to its promoter region. Thus, the two splicing variants of XBP1, XBP1-u and XBP1-s, exert completely different and specific mechanisms in regulating p53 family members: XBP1-u regulates p53 post-translationally by destabilizing its protein, whereas XBP1-s suppresses TAp73 transcriptional activity. The functional differences of the two isoforms of XBP1 are most plausibly due to their distinct C-terminal, as 26 bp of nucleotides in XBP1-u are excised during splicing, causing a codon shift in XBP1-s [4,5]. Together with the fact that the levels of both XBP1-u and XBP1-s increase in tumor tissues [4,8], our results strongly suggest that both of them are crucial for tumorigenesis by exerting their
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specific functions in suppressing p53 and TAp73. In conclusion, in this study we unraveled a novel role of XBP1-s in regulating TAp73, as well as the importance of XBP1-s/TAp73 axis in promoting colorectal cancer cell proliferation and colony formation. Our findings not only provide novel insights regarding the aberrant TAp73 regulation in tumors, but also link up tumor cells ER stress with the tumor suppressive activity of TAp73. Moreover, these findings also suggest the potential of targeting XBP1-s/TAp73 axis for treating cancers.
[10]
Conflicts of interest
[11]
The authors declare no conflict of interest. Acknowledgements This work was supported by the grants from the Natural Science Foundation of Chongqing No. cstc2018jcyjAX0411 and cstc2018jcyjAX0374, and the Graduate Scientific Research and Innovation Foundation of Chongqing (CYB17039). We thank Dr Peng Jiang (Tsinghua University, China) for providing TAp73 overexpression vector; and Dr Massimo Levrero (University of Rome ‘La Sapienza’, Rome, Italy) for providing long TAp73 reporter.
[7]
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Appendix A. Supplementary data [18]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.112. Transparency document
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Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.112.
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Please cite this article as: H. Ji et al., XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.112