Nad(P) Dependent Steroid Dehydrogenase-Like, Involved In Cholesterol Biosynthesis, Regulates Proliferation And Metastasis In Breast Cancer

Background Cholesterol biosynthesis pathway is a common upregulated pathway in breast cancer. NSDHL (NAD(P) Dependent Steroid Dehydrogenase-Like), a gene which is involved in cholesterol biosynthesis has not been elucidated for its role in breast cancer. Methods After knock-down of NSDHL in human breast cancer cell lines, cell proliferation assay, cell cycle analysis, 3D culture, clonogenic assay, migration and invasion assay, and wound healing assay were performed. We tested erlotinib as a target drug for NSDHL. Xenograft was developed with MDA-MB-231 transfected with NSDHL shRNA in NOD/SCID mice. Public gene expression databases were used for the analysis of prognostic role of NSDHL in breast cancer Results Both NSDHL mRNA and protein levels were higher in a Luminal (MCF7) and triple negative cells (MDA-MB-231, BT-20) than normal breast cancer cell (MCF10A). Silencing of NSDHL by siRNA inhibited cell cycle and proliferation, and also inhibited migration and invasion in MDA-MB-231 and BT-20 cells (p< 0.05). The sensitivity to erlotinib was signicantly decreased in siNSDHL treated cells, especially in MDA-MB-231 (p<0.01). Additionally, we found that NSDHL regulated total cholesterol levels in breast cancer cells. In vivo xenograft model, NSDHL knock down in the cancer cell decreased the tumor growth and metastasis. Clinically, higher NSDHL expression in the tumor of 3951 breast cancer patients was associated with signicantly lower recurrence-free survival. Conclusions In conclusion, we showed that NSDHL might have a role for the progression of breast cancer. Further studies are needed to clarify that NSDHL can be a therapeutic target in breast cancer. and Cholesterol Esterase in 1X Assay Diluent. Determine the cholesterol concentration of the samples with the equation obtained from the linear regression analysis of the standard curve.

treated cells, especially in MDA-MB-231 (p<0.01). Additionally, we found that NSDHL regulated total cholesterol levels in breast cancer cells. In vivo xenograft model, NSDHL knock down in the cancer cell decreased the tumor growth and metastasis. Clinically, higher NSDHL expression in the tumor of 3951 breast cancer patients was associated with signi cantly lower recurrence-free survival. Conclusions In conclusion, we showed that NSDHL might have a role for the progression of breast cancer. Further studies are needed to clarify that NSDHL can be a therapeutic target in breast cancer.

Background
Breast cancer is common malignancy of women in Korea [1,2] and the major cause of mortality in females worldwide [3]. Although various progress in early diagnosis, radiation therapy and various combination of surgery improved the prognosis of breast cancer patients [4], the high mortality rates are remained. Furthermore, the identification of novel therapeutic targets is important to discover the potential biomarkers for early diagnosis and prognosis in breast cancer patients.
In previous study, using Whole-exome sequencing in 120 breast tumor and normal paired tissues, they detected 11,684 putative somatic mutations in 7,373 genes. After selected 1,116 genes with 3 or more mutations, 695 genes were chosen using messenger RNA (mRNA) expression in the whole transcriptome sequencing data. Finally, 198 somatic mutations of 50 genes were selected in 64 patients. In this result, NSDHL (NAD(P) Dependent Steroid Dehydrogenase-Like) has 3 mutations and high level of hazard ratios for recurrence using K-M plotter (Hazard Ratio = 1.43, 95% CI = 1.28 -1.61, P-value < 0.001) [5].
NSDHL (NAD(P) Dependent Steroid Dehydrogenase-Like) is a protein coding gene. Also, localized in the endoplasmic reticulum and involved in cholesterol biosynthesis [6]. Obesity is considered important risk factor in cancer [7][8][9][10] but the cause is not elucidated.
In the present study, in breast cancer cells, NSDHL expression of both mRNA and protein levels in MCF7, MDA-MB-231 and BT-20 cells were higher than non-tumorigenic epithelial cell (MCF10A). Among them, we inhibited NSDHL expression using small interfering RNA (siRNA) and studied proliferation and migration potential in breast cancer cells related with EGFR and cholesterol pathway. Moreover, we found that NSDHL regulated total cholesterol levels. After then, the tumor growth and metastasis were confirmed in NOD scid gamma mice. Clinically, we identified Patients with high NSDHL expression in total of 3951 breast cancer patients showed unfavorable outcomes on recurrence free survival (RFS) rates. Taken together, these results demonstrated that NSDHL is pivotal for the tumorigenesis in breast cancer.

Expression of NSDHL in breast cancer cell lines
In order to identify the function of NSDHL, the NSDHL mRNA and protein levels were  Table 4 and Figure 5A, B, and C, Erlotinib could inhibit cell proliferation in a dose-dependent manner. In detail, the IC50 value in MCF (-74.07, + 46.42), MDA-MB-231 (-83.54, + 27.62) and BT-20 (-168.2, + 78.28) cells.
Furthermore, the highest sensitivity was found in siCONTROL of BT-20 cell ( Figure 5C) and the difference between siCONTROL and siNSDHL was largest in MDA-MB-231 cell ( Figure   5B). The sensitivity of Erlotinib was significantly lower in siNSDHL treated cells, especially in MDA-MB-231 (p<0.01). Also, the extent of proliferation is consistent with the degree of knock-down levels in protein of EGFR and CDK2 ( Figure 8B). In these results indicated that Erlotinib effectively reduced transfected NSDHL siRNA in breast cancer cells in relative to EGFR pathway.

Downregulation of NSDHL inhibits MDA-MB-231 and BT-20 cells in the migration and Invasion
Since cancer cell migration play very critical roles in cancer metastasis. Thus, we  Figure   6B). Consistently, the wound healing assay also showed that the migration rate was decreased by silencing of NSDHL in TNBC cells ( Figure 6C). In detail, compared with the control cell groups, scratch wound reduced differently for 24 h, especially in MDA-MB-231 cells (p<0.05). In western blotting assay, we found that binding to specific sterol element, SREBP-1 and LDLR expressions were reduced in siRNA transfected cells ( Figure 8B). Therefore, these data indicated that the pivotal role of NSDHL on the migration and invasion in TNBC cells.  Figure 7B). To support these results, we analyzed in western blotting with involved in sterol biosynthesis enzymes. In western blotting assay, we found that binding to specific sterol element, EGFR, SREBP-1 and LDLR expressions were reduced in siRNA transfected cells ( Figure 8B).
These results were definitely confirmed that NSDHL involved in the molecular mechanisms might be associated with cholesterol in breast cancer cells.

Predicted Mechanism of NSDHL regulation in breast cancer cells
To determine the biological significance of NSDHL in breast cancer cells related with EGFR/ biosynthesis pathway of cholesterol, we confirmed by western blotting analysis ( Figure 8A, B). In these results, silencing of NSDHL decreased whole protein expressions involved in EGFR/biosynthesis pathway related with proliferation and migration.

Downregulation of NSDHL expression in MDA-MB-231 cells by shRNA Lentiviral Particles Transduction
We performed short hairpin RNA (shRNA) (h) Lentiviral Particles Transduction in MDA-MB-231 for in vivo study. Selected stable clones expression the shRNA via Puromycin dihydrochloride selection were measured mRNA expression levels by RT-qPCR (p<0.01) and protein levels in western blot analysis ( Figure 9A, B). These results supported that the suppression of NSDHL by shRNA could lead to inhibition of MDA-MB-231 cell progression and metastasis in NSG mice.
Tumor progression and lung metastasis of NSG mouse was decreased by shNSDHL in in vivo NOD scid gamma mice (NSG mice) born in between july 31, 2018 and august 9, 2018 injected on september 21, 2018. 5 shCONTROL mice and 5 shNSDHL mice were injected into each left breast ( Figure 10A). Tumor growth in NSG mouse was significantly inhibited by NSDHL knock-downed cell for 44 day (p<0.01) ( Figure 10B). Also, the control and knock-down tumor were different in volume, weight (p<0.01) and immunohistochemistry ( Figure 10C, D and E).

Clinical implication of NSDHL in breast cancer
As shown in Table 2

Discussion
NSDHL (NAD(P)-dependent steroid dehydrogenase-like) play a critical role in meiosis activation [23]. In the skin, MASs activate because of the skin abnormalities caused by cholesterol synthesis in human and animal, including SC4MOL (sterol-C4-methyl oxidase-like 1) [15,24] and EGFR may be important in these skin changes in human and animal through EGFR signaling [11,15,25]. In addition, inactivation of SC4MOL and NSDHL reduced EGFR expression. In many studies, they implicated the function of the cholesterol pathway in tumor grow and response to treatment [15]. For instance, , the sensitivity of neck and head cancer cells in apoptosis [26,27] and through EGFR signaling, sterol composition of the membrane is regulated [15,28]. Furthermore, NSDHL derived from ER membranes [18], cooperate with lipid rafts to promote metastasis by lipid-depleted serum (LDs) [22]. However, the impact of NSDHL on progression and metastasis of breast cancer remained and underlying mechanism is not clearly demonstrated.
We here use a NSDHL, which was selected as a therapeutic target, function in breast cancer in in vitro, in vivo and clinal strategy. Our data imply a important functional role for NSDHL in breast cancer and potential biomarker for diagnosis in breast cancer patients.
From whole-exome sequencing in 120 breast tumor and normal paired tissues, NSDHL has 3 mutations and high level in hazard ratio [5].  [29]. Also, the PI3K/AKT/mTOR pathway, implicated in endocrine resistance, is a major intracellular pathway, which leads to cell proliferation [30,31]. LDL-cholesterol induced breast cancer cell growth, migration [32] and LDLR accelerates LDL-cholesterol that increased recurrence and mortality in breast cancer [33]. We analyzed the regulation of NSDHL and EGFR/AKT/LDLR expressions in protein level and found through this pathway that affected cell growth.
Erlotinib, a small-molecule epidermal growth factor receptor tyrosine kinase inhibitor has potent effect in non-small-cell lung cancer proliferation In that results, H322 NSCLC cells highly expressed EGFR was accompanied by G1/S phase arrest, because of the cell growth inhibition by erlotinib [34]. And regardless of EGFR expression, the ability of erlotinib to inhibit CDK2 activity is important role for cellular sensitivity to erlotinib and the p27 expression in the cytoplasm also involved in erlotinib resistance [35]. Additionally, depletion of SC4MOL and NSDHL, sensitizes tumor cells to EGFR inhibitor [15]. Therefore, we tested the sensitivity of Erlotinib, a small-molecule EGFR tyrosine kinase inhibitor, related with NSDHL in breast cancer cells. These results also indicated that erlotinib is effectively reduced breast cancer cell growth with NSDHL relation to CDK2 protein expression.
In addition, NSDHL has impact on metastasis in TNBC cell lines. Specifically, in NSDHL induced cells, migration and invasion ability were increased consistent with the degree of wound closer. In previous study, SREBP-1 involved in cholesterol pathway promotes migration and invasion in breast cancer [36]. NSDHL translocated to the plasma membrane from the intracellular compartment and promote metastasis relation to LDLR expression relation to LDL-cholesterol [22,32]. We found that regulated NSDHL reduced SREBP-1 and LDLR expression of protein level in breast cancer cells.
We were concerned about the amount of cholesterol in breast cancer, total cholesterol levels were measured in the cells, resulting in a high cholesterol level in order of BT-20, MDA-MB-231 and MCF7 cells. After then, we are going to consider the relationship between EGFR, cholesterol and breast cancer in relation to the amount of cholesterol expressed by EGFR.
To reveal the mechanism of NSDHL in cholesterol pathway, we analyzed various enzymes, involved in cholesterol pathway. For example, EGFR, AKT, SREBP-1 and LDLR [37]. As well as we tested CDK2. In previous study, the LDLR expression is stimulated by activated SREBP-1 and in cholesterol homeostasis regulation [38] and SREBP-SCAP complex to move to the Golgi is promoted by activated EGFR/mTOR signaling [37]. In these results, we found out NSDHL regulated by EGFR/AKT pathway. And we predicted that NSDHL affected to cell cycle and proliferation from CDK2 and translocated to LDLR related with cholesterol. But, in migration and invasion, NSDHL was affected by SREBP-1 relation to LDLR.
After then, we confirmed the progression and metastasis ability of NSDHL in NSG mice.
The downregulation of NSDHL induced a decrease in tumor growth and NSDHL expression through immunohistochemistry. Also, metastasis in Hematoxylin and eosin staining (H&E staining) of lung was inhibited compared with control cell.
Finally, the Cox Proportional Hazard Ratio Model Analysis in 3951 breast cancer patients revealed that NSDHL is a significantly meaningful gene in breast cancer clinically. Page 17/33 Therefore, these results indicated that NSDHL is a pivotal molecule for prognosis in breast cancer.

Conclusion
In conclusion, NSDHL is critical for de novo progression and metastasis in breast cancer cells and NSG mice. Therefore, we indicated that NSDHL is novel therapeutic targets [22,39]