The effect of DDIT3 on luminal A type breast cancer

doi:10.21203/rs.3.rs-3624582/v1

Purpose: To analyze the phenotypic changes of breast cancer (BC) cell before and after DDIT3 knockdown/overexpression, and preliminarily explore the regulatory mechanism. Also, to analyze the relationship between DDIT3 and prognosis by combining with bioinformatics methods, which provide a basis for further research on DDIT3 targeted treatment of BC.

Methods: Loss- and gain-of-function studies, DDIT3 in MCF-7 (luminal A), and RNA-seq analysis were employed to investigate the functional impact of DDIT3 on BC cell proliferation and drug resistance. The relationship between DDIT3 and the prognosis of BC patients was systematically assessed using the tissue microarray technique along with qRT-PCR and publicly available data.

Results: Survival analysis showed that patients with lower DDIT3 expression in luminal A type BC or BC patient which were undergoing endocrine therapy had a poorer prognosis, and DDIT3 expression was associated with overall survival (OS) significant. Following the knockdown of DDIT3 in MCF-7 cells, the proliferation rate was significantly increased, and drug resistance ability was just reversed. On the contrary, overexpression of DDIT3 had a relative inhibitory effect on target cell proliferation. Notably, the inhibition of DDIT3 expression upregulated TP63 and downregulated PDGFR, with the results being exactly opposite after the overexpression of DDIT3.

Conclusion: These results have revealed that DDIT3 plays a critical role in luminal A type BC cell proliferation and TAM resistance, and it holds potential prognostic value in BC. Overall, DDIT3 may exert its functions in luminal A type BC by modulating the expression of TP63 and PDGFR.

DDIT3

breast cancer

cell proliferation

drug resistance

prognosis

BC is the most common malignant tumor in females (Xu Y et al. 2023). According to the Global Cancer Statistics 2020, there were approximately 2.3 million new cases of BC, and its overall incidence (11.7%) has surpassed lung cancer (11.4%) to become the leading cause of cancer worldwide (Sung H et al. 2021). Currently, chemotherapy remains the primary clinical treatment approach for BC, and TAM is the most widely used first-line chemotherapy drug in the clinic to treat estrogen receptor-α positive BC (Kerr AJ et al. 2022; Rajendran S et al. 2021). Advances in understanding the molecular basis of BC development are attributed to genetic and epigenetic analysis of oncogenes and tumor suppressor genes (Nolan E et al. 2023; Sukocheva OA et al. 2022). It is believed that loss-of-function mutations in tumor suppressor genes and gain-of-function mutations in proto-oncogenes can trigger cancer development (Wang LH et al. 2018; Wang H, et al. 2006; Guérit E et al. 2021). Additionally, epigenetic changes in the genome can lead to abnormal transcription and further promote the malignant transformation of tumors (Garcia-Martinez L et al. 2018). Despite an improved understanding of these tumor characteristics, the pathogenesis of BC remains unclear, and specific targeted drugs are still lacking. Therefore, the discovery of additional genes that may regulate BC proliferation may provide important insights for the development of new drugs and clinical treatments.

As a cellular stress transcription factor, DDIT3 is generally widely expressed at an undetectable level when induced by cellular stress under normal conditions (Oyadomari S and Mori M 2004). However, it is typically overexpressed and mediates apoptosis under various physiological or stressful conditions, such as ER stress, DNA damage, growth arrest, and hypoxia (Wang M et al. 2016; Chen X et al. 2023). Numerous studies have confirmed that DDIT3-mediated apoptosis is involved in many human diseases, such as neurodegenerative diseases, diabetes, and metabolic diseases (Liu Y et al. 2021; Duan H et al. 2016; Mercuri E et al. 2020; Hu H et al. 2019; Li Y et al. 2014). Additionally, as a regulatory factor of nuclear transcription, DDIT3 can also control the expression of numerous stress response genes involved in cell processes like apoptosis, autophagy, and proliferation at the transcriptional level (Gao J et al. 2021; Almanza A et al. 2019). DDIT3 can induce or inhibit apoptosis in different tumors or under different conditions. This variability is due to differences in tumor and disease backgrounds, and stress intensity, and duration, and cell behaviors induced by DDIT3, resulting in differing outcomes (Suzuki K et al. 2017; Rozpedek W et al. 2016; Pavlović N et al. 2021; Wang Z et al. 2019; Patil N et al. 2014). However, it is undeniable that DDIT3 is a double-edged sword, serving as both a cell death mechanism and a factor that can prolong tumor survival time. Consequently, the focus of future research is expected to involve the discovery or design and synthesis of anticancer therapies or drugs that can induce ER stress, enhance DDIT3 expression, stimulate its pro-apoptotic function, or inhibit its pro-cancer function. Furthermore, identifying key regulators that can achieve both aims is one of the ultimate research goals.

Cell culture, viability assay and stress treatment

HEK293T and human breast cancer (BC) cell lines (MCF-7) were obtained from the National Infrastructure of Cell Line Resource. HEK293T cells were grown in DMEM medium (Hyclone), while MCF-7 cells were cultured in α-MEM medium (Hyclone) supplemented with 10% FBS (Ausbian) and 10 µg/mL insulin. All the cells were cultured at 37°C in the presence of 5% CO₂. Cells were detached using a 0.25% Trypsin-EDTA solution (Hyclone).

Transfection and quantitative RT-PCR

The SiDDIT3-3 (5'-GGT CCT GTC TTC AGA TGA A-3') and SiDDIT3-7 (5'-CTG ATT GAC CGA ATG GTG A-3') were designed. Cell transfection was achieved using jetPRIME® transfection reagent (Polyplus, France). 100 nM SiRNA was added to 4×10⁵ MCF-7 cells that had already been seeded and adhered to a 6-well plate. After incubation for 48 hours, total RNA was extracted using Trizol reagent. RNA was reverse-transcribed with the PrimeScript RT kit (Takara, Japan). qPCR detection was performed using the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus, Takara, Japan) on a Real-Time System (BIO-RAD, CFX ConnectTM). The primers for qRT-PCR are listed in Table 1. Data were analyzed using the 2^−ΔΔCt method.

Table 1

Primers used for quantitative PCR
Primer	Sequence (5'-3')
DDIT3-qF	CAT TGC CTT TCT CCT TCG GG
DDIT3-qR	CCA GAG AAG CAG GGT CAA GA
TP63-qF	GGA CCA GCA GAT TCA GAA CGG
TP63-qR	AGG ACA CGT CGA AAC TGT GC
PDGFR-qF	TGG CAG TAC CCC ATG TCT GAA
PDGFR-qR	CCA AGA CCG TCA CAA AAA GGC
Actin-qF	CCC AGC ACA ATG AAG ATC AA
Actin-qR	ACA TCT GCT GGA AGG TGG AC

Full length amplification of DDIT3 gene

Based on the mRNA coding region sequence of DDIT3 (NM_001195053.1) and Primer Premier 6.0 software, forward and reverse primers of DDIT3 were designed to amplify the full length of DDIT3 gene, and then recombine DDIT3 gene with the eukaryotic expression vector pIRER2. In order to correctly express the recombinant plasmid in cells, Xho I and BamH I cleavage sites were introduced at the 5'-end of the forward and reverse primers. The primer sequences were obtained as follows (the introduced cleavage site sequences and protective bases are shown in the box and parentheses).

Table 2

Primers used for *DDIT3* full-length
Primer	Sequence (5'-3')
DDIT3-Forward	5'-CCGCTCGAGCGGATGGAGCTTGTTCCAGCCAC-3'(Xho I)
DDIT3-Reverse	5'-CGCGGATCCGCGTCATGCTTGGTGCAGATTCAC-3'(BamH I)

Colony formation assay

MCF-7 cells transfected with SiRNAs (counted as 2000 cells) were resuspended in medium, added to a 6-well plate, and maintained at 37°C in the presence of 5% CO₂. Cells were replenished with fresh medium every 3 days. At the end of the assay (approximately 12 days), the cells were stained with 0.5 mL of crystal violet for 1 hour, then washed and imaged as described in the product manual. The colonies were counted and subjected to statistical analysis.

CCK-8 Cell proliferation assay

For the cell proliferation analysis, 1×10⁴ MCF-7 cells transfected with SiRNA were seeded in 96-well plates. Cell viability was determined on days 1, 3, 5, and 7 using CCK-8 kits. Absorbance at 450 nm was measured after a 2-hour incubation at 37°C. The data were normalized to day 1 and presented as mean ± standard deviation.

Cell drug resistance assay

In clinical practice, TAM has almost no anti-tumor activity, and its function mainly depends on the active metabolite 4-OH-TAM (4-OHT). Therefore, 4-OHT is administered to cells during the assay. Cells were inoculated at a density of 1×10⁴ cells per well in 96-well plates and cultured overnight at 37°C. Various concentrations of 4-OHT (ranging from 0 µM to 32 µM) were dissolved in anhydrous methanol and diluted with α-MEM medium. These concentrations were administered after MCF-7 cells adhered. After 72 hours, CCK-8 reagent was used to measure the absorbance of each well at 450 nm.

Bioinformatic analysis

The expression of DDIT3 in different cancer types, as well as between cancer samples and adjacent tissues, was analyzed using data from the GEO and Oncomine database (https://www.oncomine.org/). P-values less than 1×10^− 4 were considered statistically significant. The UALCAN dataset (http://ualcan.path.uab.edu/index.html) was used to analyze DDIT3 protein levels in breast cancer tissue. Kaplan-Meier Plot database (https://kmplot.com) and TCGA portal (http://www.tumorsurvival.org/index.html) websites were utilized to examine DDIT3 expression in BC survival. The enrichment score and multiple test corrections were used to reveal the biological characteristics and genetic regulatory network of DDIT3.

Statistical analysis

Statistical analysis all data are expressed as means ± SD. For each experiment, three independent replicates were performed. Statistical evaluation was conducted using student t-tests and by ANOVA for repeated measures (Graphpad Prism 9.5). A value of * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control was considered statistically significant.

The expression of DDIT3 was significantly correlated with estrogen receptor type and endocrine therapy in BC patients.

There are few reports on the expression of DDIT3 in BC. In our previous research on BC, we found that the expression of DDIT3 was significantly related to estrogen receptor and BC cell growth related signaling pathway. Furthermore, combining the data from the TCGA database and Kaplan-Meier plot database (Brest cancer RNA-seq, n = 2976), and analyzing existing clinical samples, we found that DDIT3 is expressed in almost all cancers (Fig. 1a). In addition, there was a negative correlation between DDIT3 and overall survival (OS) of BC patients (p = 0.001, HR = 1.49) (Fig. 1b); but in contrast, there was a positive correlation between DDIT3 and OS of BC patients (p = 0.0021, HR = 0.55) (Fig. 1c). In addition, we also statistically learned that the expression of DDIT3 was positively correlated with the OS of BC patients who received TAM treatment (p = 0.0089, HR = 0.68) (Fig. 1d), and the expression of DDIT3 was negatively correlated with the OS of BC patients who did not receive TAM treatment (p = 0.011, HR = 1.68) (Fig. 1e). It was pointed out that there was an important relationship between the expression of DDIT3, the pathological classification and TAM resistance of BC. Also, it has an important impact on the prognosis of luminal A type BC patients.

In addition, the correlation between the level of DDIT3 in BC tissue samples and the tumor type, pathological stage and lymph node metastasis was analyzed by tissue microarray technology. Combined with clinical medical records, select BC cases that are comparable in clinical staging, pathological type and treatment methods. To study the expression of DDIT3 and speculate the relationship between the level of DDIT3 and the prognosis of BC patients. We entrusted Shanghai Xinchao Biotechnology Co., Ltd. to conduct 145 tissue microarray detection of BC, and found that the expression of DDIT3 was significantly correlated with the pathological grade of BC and estrogen receptor biological characteristics of BC (p < 0.05) (Table 3). The expression of DDIT3 in BC tissue was also significantly different from that in normal breast tissue (p < 0.05) (Table 4). Therefore, we have reason to believe that DDIT3 plays an important role in the proliferation of ER positive BC.

Table 3

Correlation between DDIT3 expression and clinicopathological characteristics of BC
	variables	DDIT3 expression		total	r_s	P value
	variables	high	low	total	r_s	P value
Age (year)					0.031	0.712
	< 50	17	43	60
	>=50	25	55	80
Grade					-0.229	0.006*
	1	7	8	15
	2	35	76	111
	3	0	14	14
T stage					0.086	0.315
	T1	8	23	31
	T2	28	66	94
	T3	6	8	14
	dull			1
N stage					-0.137	0.108
	N0	21	28	49
	N1	9	36	45
	N2	9	27	36
	N3	3	5	8
	dull			2
TNM stage					-0.051	0.554
	Ι	4	7	11
	II	25	55	80
	III	13	34	47
	dull			2
ER					-0.175	0.048*
	negative	17	25	42
	positive	30	65	85
	Dull			13
PR					-0.160	0.072
	negative	20	33	53
	positive	17	57	74
	Dull			13
HER2					0.047	0.660
	negative	18	48	66
	positive	8	17	25
	Dull			49
*p < 0.05

Table 4

Expression of DDIT3 in BC tissues and adjacent tissues
	n	DDIT3 expression		Chi-square Value	P Value
		High(%)	Low(%)	4.2593	0.03903694*
Breast cancer	69	20	49
Breast tissues	69	10	59
*p < 0.05

To determine the correlation between DDIT3 and BC, we analyzed the immunohistochemical results of 140 cases of BC tissue and adjacent tissue microarray. This analysis confirmed that DDIT3 was highly expressed in BC tissue but not in adjacent tissue. In BC cells, DDIT3 exhibited high expression in the nucleus with a small amount of expression in the cytoplasm (Fig. 2).

DDIT3 knockdown promotes MCF-7 cell proliferation and inhibits cell 4-OHT resistance

To confirm the function of DDIT3 in BC, we employed siRNAs to silence DDIT3 expression in MCF-7 cells (Fig. 3a). The cell proliferation assay confirmed that DDIT3 knockdown can significantly promote the proliferation of MCF-7 cells (Fig. 3b). Similar to the results of the cell proliferation assay, the cell cloning assay confirmed that the clonogenic ability of target cells was significantly improved after DDIT3 knockdown (Fig. 3c, Fig. 3d). Furthermore, after the target cell DDIT3 knockdown, the cell survival rate decreased to 50% when the 4-OHT administration concentration ranged between 8–10 µM, while the cell survival rate in the SiNC group only decreased by half when the administration concentration was 16 µM (Fig. 3e). In addition, there were no significant changes in the migration ability, invasion ability, and cell cycle of target cells after DDIT3 knockdown. Taken together, we can conclude that DDIT3 has a negative regulatory effect on the proliferation of MCF-7 cells, and the drug resistance of target cells was also significantly reduced after DDIT3 knockdown.

DDIT3 overexpression inhibits BC cell proliferation

Based on the changes in the phenotype of MCF-7 cells after the knockdown of DDIT3, furthermore, we explored whether there would be precisely the opposite change in the phenotype of MCF-7 cells after the overexpression of DDIT3 (Fig. 4a, Fig. 4b). We determined that overexpression of DDIT3 relatively inhibits cell proliferation, but the inhibitory effect was relatively weakened compared to DDIT3 knockdown (Fig. 4c), and its overexpression has no significant effect on 4-OHT resistance in MCF-7 cell lines (Fig. 4d). However, slightly different was that after overexpression of DDIT3, the colony formation ability of MCF-7 suddenly decreases. After repeated experiments with 3 transfection reagents, 4 eukaryotic expression vectors and four different types of BC cells, we found that after overexpression of DDIT3, all the experimental group cells died in large numbers and their ability of colony formation declined sharply. Gradually increase the number of cells laid in the 6-well plate to 5×10³ cells per well, and incubation 14 days, the control group were able to form a large number of cell clusters, while the overexpression group cells in all parallel wells were unable still. Similarly, due to the significant death of target cells after overexpression of DDIT3, experiments such as cell migration, cell invasion, and cell cycle that require a certain number of cells to complete cannot proceed smoothly.

DDIT3 regulates the proliferation and TAM resistance of Luminal A type BC cells through regulate the mRNA expression of TP63 and PDGFR.

In order to further explore the biological function of DDIT3, we conducted RNA-Seq experiments on DDIT3 knockdown MCF-7 cells. It was confirmed that DDIT3 was associated with the proliferation and TAM resistance of MCF-7 cells. Therefore, we screened dozens of genes with significant differential expression related to cell proliferation and drug resistance from the RNA-Seq results and conducted qRT-PCR assays for validation. Ultimately, we speculated that DDIT3 can negatively regulate the mRNA expression of the TP63 gene, affecting the p53 signaling pathway, and then regulating the proliferation of BC cells (Fig. 5a). In addition, DDIT3 may function by the positive regulation of PDGFR mRNA expression, and then regulate the TAM resistance of breast cancer cells through the EGFR tyrosine kinase inhibitor resistance signaling pathway (Fig. 5b).

DDIT3 is a transcription factor induced by various adverse physiological conditions, including endoplasmic reticulum stress (Berastegui N et al. 2022). It participates in biological processes such as cell growth arrest, nutrient deprivation, hypoxia, genotoxicity, and cell differentiation (Tolomeo M et al. 2020). Moreover, it plays a role in the occurrence, development, and prognosis of various tumors (Liu Y et al. 2022; Zhang Y et al. 2019; Xu K et al. 2019). Many studies have confirmed that DDIT3-induced cell apoptosis is significant in various cancer processes (Mishra A et al. 2021; Iino Y et al. 1997; Misir S et al. 2023). DDIT3 can induce or suppress cell apoptosis in different tumors or under varying ER stress conditions (Suzuki K et al. 2017; Patil N et al. 2014). These differences are attributed to the diverse backgrounds of tumors and diseases, the intensity and duration of stress, and the distinct cellular responses induced by DDIT3, resulting in varying outcomes. Therefore, whether it functions as a proapoptotic factor or a protective factor remains to be defined in specific cell types.

Although the efficacy of TAM in the treatment of estrogen receptor-α positive BC has been recognized and widely used in clinical practice, in the clinical application of TAM, in addition to some patients who have been resistant to TAM, about 40% of patients will inevitably develop acquired drug resistance (Mishra A et al. 2021). Even under the condition of conventional use of TAM, clinical manifestations such as tumor recurrence and metastasis still occur, which seriously increases the mortality of BC (Iino Y et al. 1997). The emergence of TAM resistance is not only detrimental to the prognosis of patients, but also increases the damage caused by adverse drug reactions (Misir S et al. 2023). Therefore, how to reduce the TAM resistance of BC, and then improve the drug efficacy, is a hot issue to be solved urgently in clinical treatment of BC.

In this report, we confirmed that the expression of DDIT3 was significantly related to the estrogen receptor subtype, tumor grade, and OS of luminal A type BC patients through statistical analysis. The expression of DDIT3 in breast cancer tissues and adjacent tissues was also significantly different. We observed that after the knockdown of DDIT3 in MCF-7 cells, the proliferation rate, drug resistance (4-OHT), and clonogenic ability were significantly increased. Overexpression of DDIT3 relatively inhibited the proliferation of target cells, but the inhibitory effect was slightly different from knockdown, and its overexpression had no significant effect on drug resistance in MCF-7 cell lines. To further explore the biological function of DDIT3, we conducted RNA-Seq experiments on DDIT3 knockdown MCF-7 cells. It was confirmed that DDIT3 was associated with the proliferation and TAM resistance in MCF-7 cells. Therefore, we screened dozens of genes with significant differential expression related to cell proliferation and drug resistance from the RNA-Seq results and conducted qRT-PCR assays for validation. Ultimately, we speculated that DDIT3 can negatively regulate the mRNA expression of the TP63 gene, affecting the p53 signaling pathway, and then regulating the proliferation of BC cells. In addition, DDIT3 may function by positively regulating PDGFR mRNA expression and, consequently, regulating the TAM resistance of BC cells through the EGFR tyrosine kinase inhibitor resistance signaling pathway.

DDIT3 is a double-edged sword that not only serves as a cell death mechanism but also prolongs tumor survival time. Therefore, discovering or synthesizing anticancer therapies or drugs that could induce ER stress, promote DDIT3 expression, stimulate its pro-apoptotic function, or block its pro-cancer function would inevitably be the focus of future research. Alternatively, finding key regulatory factors that could achieve the ultimate goals. We speculate that the PERK-eIF2α-ATF4-DDIT3 pathway is highly likely to be the key path to achieving the ultimate goal. Firstly, the PERK-eIF2α-ATF4-DDIT3 pathway is one of the main pathways of the unfolded protein response (Wang Z et al. 2019; Rozpedek W et al. 2016). Secondly, in the most inevitable ER stress state in tumors, high expression of DDIT3 downregulates the expression of Bcl-2, increases the translocation of Bax from the cytoplasm to the mitochondria, and triggers cell apoptosis. The sustained PERK signal inhibits cell proliferation and promotes cell apoptosis (Lin JH et al. 2009). Furthermore, even when the ER stress state is partially corrected, PERK can phosphorylate eIF2α, activate ATF4, and induce DDIT3 expression (Xu L et al. 2021). Summarizing the above regulatory results, it can be concluded that in the PERK-eIF2α-ATF4-DDIT3 pathway, we can form the desired reciprocating cycle to achieve the ultimate goal. Unfortunately, all current data tends to be more theoretical, and in fact, the various factors in the PERK-eIF2α-ATF4-DDIT3 pathway can be regulated or influenced by multiple factors, and the specific efficacy of this pathway in different tumors is not yet known. There are currently no relatively suitable and accurate theoretical research results that can be used for reference. However, it is precisely because of this that further research on the function and mechanism of DDIT3 in tumors under endoplasmic reticulum stress may provide important new insights for the pathogenesis of tumors and clinical drug research.

DDIT3, as a potential therapeutic target for tumors, is also receiving increasing attention, necessitating more detailed in vivo studies to evaluate its efficacy. However, so far, most of the research is still in the laboratory, in vitro cell stage (Kalimuthu K et al. 2021; Chiu CS et al. 2019; Xiao F et al. 2021). In addition, research on DDIT3 provides lots of creative ideas for the screening of key regulatory factors that indirectly or directly induce cell apoptosis and the improvement of their mechanisms of action (Xu T et al. 2019; Chen P et al. 2016; Ge J et al. 2019). However, there are still many questions to be answered regarding the role of DDIT3 in tumors, such as how these regulatory molecules interact, which one plays a decisive role, and whether the ongoing regulatory network is correct. With the deepening of research, the function and molecular mechanism of DDIT3 in oncology will continue to be revealed, and DDIT3 or DDIT3 key regulatory factor-targeted treatment plans will also find new directions for cancer clinical treatment.

Our study provides evidence that DDIT3 is critical for BC cell proliferation and TAM resistance, and it has the potential prognostic value in BC. DDIT3 may achieve its functions in luminal A type BC by modulating TP63 and PDGFR mRNA expression.

Funding

This work was supported by funds from the Natural Science Foundation of Heilongjiang Province (Grant Number LH2021H056); Pre-research project of Heilongjiang Academy of Sciences (Grant Number YY2022GJS01); Research Business Projects of Provincial Scientific Research Institutes in Heilongjiang Province (Grant Number ZNBZ2022GJS04).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Guoqing Huang, Jiaying Xue, Difei Cao. The first draft of the manuscript was written by Guoqing Huang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Consent to publish

The authors affirm that all research participants provided informed consent for publication of this manuscript.

Almanza A, Carlesso A, Chintha C et al (2018) Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications. FEBS J 286(2):241-278. doi: 10.1111/febs.14608.
Berastegui N, Ainciburu M, Romero JP et al (2022) The transcription factor DDIT3 is a potential driver of dyserythropoiesis in myelodysplastic syndromes. Nat Commun 13(1):7619. doi: 10.1038/s41467-022-35192-7.
Chen P, Hu T, Liang Y et al (2016) Neddylation Inhibition Activates the Extrinsic Apoptosis Pathway through ATF4-CHOP-DR5 Axis in Human Esophageal Cancer Cells. Clin Cancer Res 22(16):4145-4157. doi: 10.1158/1078-0432.CCR-15-2254.
Chen X, Shi C, He M, Xiong S, Xia X (2023) Endoplasmic reticulum stress: molecular mechanism and therapeutic targets. Signal Transduct Target Ther 8(1):352. doi: 10.1038/s41392-023-01570-w.
Chiu CS, Tsai CH, Hsieh MS,et al(2019) Exploiting Honokiol-induced ER stress CHOP activation inhibits the growth and metastasis of melanoma by suppressing the MITF and β-catenin pathways. Cancer Lett 442:113-125. doi: 10.1016/j.canlet.2018.10.026.
Duan H, Arora D, Li Y, et al (2016) Identification of 1,2,3-triazole derivatives that protect pancreatic β cells against endoplasmic reticulum stress-mediated dysfunction and death through the inhibition of C/EBP-homologous protein expression. Bioorg Med Chem 24(12):2621-2630. doi: 10.1016/j.bmc.2016.03.057.
Gao J, Liu Y, Ji J, Liu Z (2021) [Heat stress induces neuronal apoptosis by up-regulating endoplasmic reticulum stress pathway]. Nan Fang Yi Ke Da Xue Xue Bao 41(5):702-709. Chinese. doi: 10.12122/j.issn.1673-4254.2021.05.10.
Garcia-Martinez L, Zhang Y, Nakata Y, Chan HL, Morey L (2021) Epigenetic mechanisms in breast cancer therapy and resistance. Nat Commun 12(1):1786. doi: 10.1038/s41467-021-22024-3.
Ge J, Sun H, Li J, et al (2019) Involvement of CHOP in activin A‑induced myeloma NS‑1 cell apoptosis. Oncol Rep 42(6):2644-2654. doi: 10.3892/or.2019.7382.
Guérit E, Arts F, Dachy G, Boulouadnine B, Demoulin JB (2021) PDGF receptor mutations in human diseases. Cell Mol Life Sci 78(8):3867-3881. doi: 10.1007/s00018-020-03753-y.
Hu H, Tian M, Ding C, Yu S (2019) The C/EBP Homologous Protein (CHOP) Transcription Factor Functions in Endoplasmic Reticulum Stress-Induced Apoptosis and Microbial Infection. Front Immunol 9:3083. doi: 10.3389/fimmu.2018.03083.
Iino Y, Maemura M, Morishita Y (1997) Mechanism of acquired antiestrogen resistance and its management in breast cancer. Nihon Rinsho 55(5):1149-1154. Japanese.
Kalimuthu K, Kim JH, Park YS, et al (2021) Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity. J Cell Physiol 236(9):6666-6677. doi: 10.1002/jcp.30329.
Kerr AJ, Dodwell D, McGale P, et al (2022) Adjuvant and neoadjuvant breast cancer treatments: A systematic review of their effects on mortality. Cancer Treat Rev 105:102375. doi: 10.1016/j.ctrv.2022.102375.
Li Y, Guo Y, Tang J, Jiang J, Chen Z (2014) New insights into the roles of CHOP-induced apoptosis in ER stress. Acta Biochim Biophys Sin (Shanghai) 46(8):629-640. doi: 10.1093/abbs/gmu048.
Lin JH, Li H, Zhang Y, Ron D, Walter P (2009) Divergent effects of PERK and IRE1 signaling on cell viability. PLoS One 4(1):e4170. doi: 10.1371/journal.pone.0004170.
Liu Y, Chen DQ, Han JX, Zhao TT, Li SJ (2021) A Review of Traditional Chinese Medicine in Treating Renal Interstitial Fibrosis via Endoplasmic Reticulum Stress-Mediated Apoptosis. Biomed Res In 2021:6667791. doi: 10.1155/2021/6667791.
Liu Y, Liang X, Zhang H, et al (2022) ER Stress-Related Genes EIF2AK3, HSPA5, and DDIT3 Polymorphisms are Associated With Risk of Lung Cancer. Front Genet 13:938787. doi: 10.3389/fgene.2022.938787.
Mercuri E, Lucibello S, Perulli M, et al (2020) Longitudinal natural history of type I spinal muscular atrophy: a critical review. Orphanet J Rare Dis 15(1):84. doi: 10.1186/s13023-020-01356-1.
Mishra A, Srivastava A, Pateriya A, et al (2021) Metabolic reprograming confers tamoxifen resistance in breast cancer. Chem Biol Interact 347:109602. doi: 10.1016/j.cbi.2021.109602.
Misir S, Yaman SO, Petrović N, et al (2023) circRNAs in drug resistance of breast cancer. Oncol Res. 30(4):157-172. doi: 10.32604/or.2022.027547.
Nolan E, Lindeman GJ, Visvader JE (2023) Deciphering breast cancer: from biology to the clinic. Cell 186(8): 1708-1728. doi: 10.1016/j.cell.2023.01.040.
Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 11(4):381-9. doi: 10.1038/sj.cdd.4401373.
Patil N, Ahmed Kabeer Rasheed S, Abba M, et al. A mechanistic study on the metastasis inducing function of FUS-CHOP fusion protein in liposarcoma. Int J Cancer. 2014 Jun 15;134(12):2808-19. doi: 10.1002/ijc.28638.
Pavlović N, Heindryckx F. Exploring the Role of Endoplasmic Reticulum Stress in Hepatocellular Carcinoma through mining of the Human Protein Atlas. Biology (Basel). 2021 Jul 9;10(7):640. doi: 10.3390/biology10070640.
Rajendran S, Swaroop SS, Roy J, et al. p21 activated kinase-1 and tamoxifen - A deadly nexus impacting breast cancer outcomes. Biochim Biophys Acta Rev Cancer. 2022 Jan;1877(1):188668. doi: 10.1016/j.bbcan.2021.188668.
Rozpedek W, Pytel D, Mucha B, et al. The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress. Curr Mol Med. 2016;16(6):533-44. doi: 10.2174/1566524016666160523143937.
Sukocheva OA, Lukina E, Friedemann M, et al. The crucial role of epigenetic regulation in breast cancer anti-estrogen resistance: Current findings and future perspectives. Semin Cancer Biol. 2022 Jul;82:35-59. doi: 10.1016/j.semcancer.2020.12.004.
Sung H, Ferlay J, Siegel RL, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021; 71(3): 209-249.
Suzuki K, Yasuda T, Watanabe K, Hori T, Kanamori M, Kimura T. Myxoid liposarcoma with cartilaginous differentiation showing DDIT3 rearrangement. Oncol Lett. 2017 Dec;14(6):6789-6794. doi: 10.3892/ol.2017.7056. Epub 2017 Sep 25.
Tolomeo M, Grimaudo S. The "Janus" Role of C/EBPs Family Members in Cancer Progression. Int J Mol Sci. 2020 Jun 17;21(12):4308. doi: 10.3390/ijms21124308.
Wang H, Han H, Mousses S, Von Hoff DD. Targeting loss-of-function mutations in tumor-suppressor genes as a strategy for development of cancer therapeutic agents. Semin Oncol. 2006 Aug;33(4):513-20. doi: 10.1053/j.seminoncol.2006.04.013.
Wang LH, Wu CF, Rajasekaran N, Shin YK. Loss of Tumor Suppressor Gene Function in Human Cancer: An Overview. Cell Physiol Biochem. 2018;51(6):2647-2693. doi: 10.1159/000495956.
Wang M, Kaufman RJ. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature. 2016 Jan 21;529(7586):326-35. doi: 10.1038/nature17041.
Wang Z, Yin F, Xu J, et al. CYT997(Lexibulin) induces apoptosis and autophagy through the activation of mutually reinforced ER stress and ROS in osteosarcoma. J Exp Clin Cancer Res. 2019 Jan 31;38(1):44. doi: 10.1186/s13046-019-1047-9.
Xiao F, Li H, Feng Z, et al. Intermedin facilitates hepatocellular carcinoma cell survival and invasion via ERK1/2-EGR1/DDIT3 signaling cascade. Sci Rep. 2021 Jan 12;11(1):488. doi: 10.1038/s41598-020-80066-x.
Xu K, Han B, Bai Y, et al. MiR-451a suppressing BAP31 can inhibit proliferation and increase apoptosis through inducing ER stress in colorectal cancer. Cell Death Dis. 2019 Feb 15;10(3):152. doi: 10.1038/s41419-019-1403-x.
Xu L, Bi Y, Xu Y, et al. Suppression of CHOP Reduces Neuronal Apoptosis and Rescues Cognitive Impairment Induced by Intermittent Hypoxia by Inhibiting Bax and Bak Activation. Neural Plast. 2021 Aug 21;2021:4090441. doi: 10.1155/2021/4090441.
Xu T, Huang C, Qi XT, et al. 2-Bromopalmitate sensitizes osteosarcoma cells to adriamycin-induced apoptosis via the modulation of CHOP. Eur J Pharmacol. 2019 Feb 5;844:204-215. doi: 10.1016/j.ejphar.2018.12.019. Epub 2018 Dec 12.
Xu Y, Gong M, Wang Y, et al. Global trends and forecasts of breast cancer incidence and deaths. Sci Data. 2023 May 27;10(1):334. doi: 10.1038/s41597-023-02253-5.
Zhang Y, Wu J, Jing H, Huang G, Sun Z, Xu S. Long noncoding RNA MEG3 inhibits breast cancer growth via upregulating endoplasmic reticulum stress and activating NF-κB and p53. J Cell Biochem. 2019 Apr;120(4):6789-6797. doi: 10.1002/jcb.27982. Epub 2018 Dec 16.

No competing interests reported.

The effect of DDIT3 on luminal A type breast cancer

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Cell culture, viability assay and stress treatment

Transfection and quantitative RT-PCR

Colony formation assay

CCK-8 Cell proliferation assay

Cell drug resistance assay

Bioinformatic analysis

Statistical analysis

Results

DDIT3 knockdown promotes MCF-7 cell proliferation and inhibits cell 4-OHT resistance

DDIT3 overexpression inhibits BC cell proliferation

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1