Control mice received same volume of DMSO diluted in corn oil. Each mice experiment was performed in triplicate and data were derived from at least 3 independent experiments. At least three repeated individual experiments were performed for each group, except where otherwise indicated. Difference between two groups was analyzed using Student t test. Our previous study has demonstrated that microRNA Mir has a pivotal role in inhibiting breast cancer initiation and progression NMT1 was significantly down-regulated after miR overexpression Fig.
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NMT1 expression was variable across different breast cell lines and did not correlate with molecular subtypes Fig. To investigate the clinical significance of NMT1, we assessed NMT1 expression in primary breast cancer tissues and adjacent normal breast tissue of 20 patients. NMT1 level was significantly higher in breast tumor tissues compared to adjacent noncancerous tissues, and was especially increased in triple-negative subtypes of breast cancer Fig.
Quantification of ALDH-positive cells in these cells right. The tumor image was shown on the right. Black arrows represent metastatic nodules in lungs. Moreover, NMT1 knockdown decreased the mammosphere formation in these cell lines Fig. NMT1 also promoted cell proliferation Figs. And NMT1 knockdown induced proliferation inhibition was due to cell cycle arrest, where the percentage of cells in G2M phase was increased Fig.
The expression of proteins responsible for G2M transition was decreased in NMT1 knockdown breast cancer cell lines Fig.
Furthermore, migration and invasion ability of cells was dramatically inhibited by NMT1 knockdown Figs. To further verify the results in vivo, we injected NMT1 knockdown cell lines into fourth mammary glands of 3-week-old to 4-week-old female nude mice. NMT1 knockdown could significantly inhibit tumor growth Fig.
And NMT1 knockdown in tumors was confirmed by immunohistochemistry and western blot Fig. S1M , S1N. Consistent with retarded tumor growth, the proportion of Kipositive cells was significantly lower in the NMT1 knockdown group Fig. Together, these results suggested knocking down NMT1 inhibited breast cancer progression and metastasis both in vitro and in vivo. Next, we searched for the possible mechanisms of how NMT1 regulating breast cancer growth and metastasis.
ER stress markers were then determined by Western blot. Ki67 positive cells and the numbers of metastatic lesions per lung section were counted right. Moreover, the decreased ability of migration and invasion of breast cancer via NMT1 knockdown was remarkably inhibited by administration of 4-PBA Figs. As shown in Figs.
Together, these results strongly support the role of ER stress as functional downstream mediators of NMT1 knockdown in breast cancer. As shown in Fig. Pathway enrichment analysis of the results showed that genes involved in mRNA metabolic process, translation initiation and ER localization were significantly enriched Fig.
This might suggest knocking down NMT1 would abrogate proper protein translation and processing in ER, and caused intracellular degraded protein increase, resulting in ER stress. And we found the level of poly-ubiquitinated proteins increased after NMT1 knockdown Fig. Previous reports have demonstrated that aggregation of ubiquitinated proteins would lead to dysfunction in many cellular pathways and intrinsic stress condition like ER stress and oxidative stress Quantification of ROS levels right.
Then the expression of NMT1 was detected by western blot. The expression of NMT1 was detected by western blot. Most importantly, the ability of cancer cell proliferation was significantly rescued after NAC treatment Fig. Taken together, these results indicated NMT1 inhibition could induce oxidative stress to modulate breast cancer initiation and progression. Then we determined whether oxidative stress had an influence on NMT1 expression. To gain further insight into the signaling pathways involved in the NMT1 knockdown mediated ER stress and oxidative stress-dependent regulation, we then performed a phosphokinase array to screen for phosphorylation of approximate 50 proteins in SUMNMT1Sh cell lines Fig.
Knocking down NMT1 significantly increased p21 expression to arrest cell proliferation Fig. JNK pathway regulates several important cellular functions including cell growth, invasion, autophagy and apoptosis And autophagy related proteins Beclin and Atg12 were elevated Fig. Thus, activation of JNK pathway mediated autophagy process in this situation.
Template showing the location of kinase antibodies spotted onto the Human Phospho-Kinase Array kit and relevant kinases were indicated by numbers left. Quantification of mean spot pixel densities of the indicated kinases right. GFP-LC3 puncta per cell was quantified right. ALDH-positive cells were quantified right. But, the effect of NMT1 knockdown on tumor invasion was not rescued might due to that the treatment time was not enough Fig.
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Next, we determined the ability of serial dilutions of cells obtained from these primary tumors to form tumors in secondary nude mice. SP treatment significantly rescued this effect Fig.
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Together, these results indicated JNK pathway activation, caused by NMT1 knockdown, was responsible for NMT1 knockdown mediated breast cancer progression delay both in vitro and in vivo. Ki67 positive cells were counted. Then JNK was detected by western blot. Then the expression of JNK was detected by western blot. And we wanted to identify the interplay between JNK and those two intercellular stresses. The schematic illustration for underlying mechanisms of how NMT1 knockdown regulating breast cancer initiation, growth and metastasis was illustrated in Fig. In this study, we demonstrate that NMT1 is capable of modulating breast cancer cell initiation and promoting cell proliferation and invasion through intracellular stress induced JNK pathway activation both in vitro and in vivo.
NMT1 has been reported to promote tumor progression partially due to its myristoylation to some famous oncoproteins, like SFK family kinases Previous reports demonstrated that Src contributed to JNK activation However, we did not see any significant changes in the phosphorylated levels of SFK family kinases via genetic inhibition of NMT1 in breast cancer cells. Unexpectedly, specific NMT1 interacting protein associated with JNK pathway could not be identified and overexpression of NMT1 did not affect any significant changes in phenotypes of breast cancer cells, such as cell proliferation and invasion.
These results indicated that a simple NMT1—substrate complex might not explain the changes in NMT1 inhibition to breast cancer.
Based on these findings, we hypothesized that targeting NMT1 would disrupt lots of protein function, and location, and abrogate normally biological process, causing great damage to breast cancer cellular homeostasis. Tumor cells must evolve by adapting to lots of stress conditions to achieve intercellular homeostasis, ultimately driving tumor progression. Manipulation of this balance might provide therapeutic approaches to eliminate cancer cells, especially for aggressive malignancy, such as triple-negative breast cancers. Previous reports have demonstrated that ER stress was essential for cancer cell initiation, proliferation, invasion and apoptosis 26 , 27 , But the role of ER stress in cancer cell proliferation has been controversial.
Collectively, these observations illustrated the complexity and dynamic of ER stress. The genetic inhibition of NMT1 triggered ER stress was persisted and prolonged in breast cancer cells, which had promising effects on many other biological aspects. Conversely, abundant ROS could cause severe ER stress, which was reported previously 32 and confirmed in our experiments. Cancer cells rely on the signaling capabilities of ROS for cell invasion, proliferation, and survival.
Importantly, if ROS levels are too high, ROS can promote anti-tumorigenic signaling and trigger oxidative stress-induced cancer cell senescence and cell death to inhibit tumor growth and metastasis It was well established that the role of JNK pathway in cancer was dependent on the stimulation type, strength and tissue specificity Whereas transient JNK activation was shown to promote cell survival, prolonged JNK activation mediated anti-tumorigenic effect Our study suggested that JNK activation promoted not apoptosis but autophagy and up-regulated downstream target p21 to prevent breast cancer progression after NMT1 knockdown.
These results provided a new mechanism of how oxidative stress abrogated breast tumor progression. But the mechanisms of targeting NMT1 to suppress cancer progression were not clearly illustrated yet. We have demonstrated that prolonged inhibition of NMT1 could cause poly-ubiquitinated proteins increase, trigger ER stress and oxidative stress, and result in JNK abnormal activation in breast cancer. Our study should allow us to better understand the mechanisms and factors involved in this process that may help to improve diagnostic and therapeutic approaches to breast cancer.
Perou, C. Molecular portraits of human breast tumours. Nature , Herschkowitz, J. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol. Koren, S. Breast tumor heterogeneity: source of fitness, hurdle for therapy. Cell 60 , — Prat, A. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer.
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Breast Cancer Res. Comparative oncogenomics identifies breast tumors enriched in functional tumor-initiating cells.
Natl Acad. Batlle, E. Cancer stem cells revisited. Ginestier, C. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1 , — Thinon, E. Global profiling of co- and post-translationally N-myristoylated proteomes in human cells. Farazi, T. The biology and enzymology of protein N-myristoylation.
Selvakumar, P. Potential role of N-myristoyltransferase in cancer. Lipid Res. Kim, S.ignamant.cl/wp-includes/55/487-como-hackear.php
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Blocking myristoylation of Src inhibits its kinase activity and suppresses prostate cancer progression. Cancer Res. N-Myristoyltransferase inhibition induces ER-stress, cell cycle arrest, and apoptosis in cancer cells. Betel, D. The microRNA. Nucleic Acids Res. Agarwal, V. Kertesz, M. The role of site accessibility in microRNA target recognition. Deng, L. MicroRNA inhibits self-renewal of breast cancer stem—like cells and breast tumor development. Mi, H. Supek, F. PLoS One 6 , e Soupene, E. We recommend that you Buy Click here to find out why Buy vs Rent: We recommend you Buy This tool helps you determine if you should buy or rent your textbooks, based on the total cost of ownership including current sell back value.
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