The Effects of CD82 Palmitoylation on the Metabolic Pathways of EGFR and c-Met in Breast Cancer Cells and their Molecular Mechanisms

Abstract Background: As a tumor metastasis suppressor, tetraspanin CD82 is reduced in many malignant tumors and often affects the composition of tumor microenvironment by changing the heterogeneity of cell membrane. EGFR or c-Met signaling pathway can regulate the metastasis ability of tumor cells and participate in the formation of tetraspanin web. The study of CD82 palmitoylation modification and metabolic pathway of tumor related molecules in tumor cells is still incomplete. This article focuses on studying the expression and distribution of EGFR and c-Met in cancer cells as well as related metabolic pathways and their molecular mechanisms after studying different palmitoylation site mutations.Methods: Western blot and immunofluorescence methods were used to detect the distribution of EGFR in breast cancer MDA-MB-231 cells after different CD82 palmitoylation site mutations. Then use the immunoprecipitation method to determine the interaction relationship between the molecules and the molecular mechanism.Results: We found that when CD82 combined with palmitoylation mutation at Cys5+Cys74 can enhance the internalization of EGFR, but has no effect on the expression and location of c-Met. When CD82 is combined with palmitoylation mutation at the Cys5+Cys74 site, with the assistance of tubulin, EGFR is internalized and strengthened by direct binding to CD82 and a large number of localizations on the recycling endosome. By forming the EGFR/CD82/Rab11a/FIP2 complex, it is metabolized through the circulation pathway, and re-expression of EGFR and CD82 on the cell membrane.Conclusions: From our results, we can demonatrate that CD82 palmitoylation mutation can change the distribution of EGFR in breast cancer cells, which may provide new ideas for breast cancer treatment.

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Recent Advances on Therapeutic Peptides for Breast Cancer Treatment

Current Protein and Peptide Science ◽

10.2174/1389203721999201117123616 ◽

2020 ◽

Vol 21 ◽

Author(s):

Samad Beheshtirouy ◽

Farhad Mirzaei ◽

Shirin Eyvazi ◽

Vahideh Tarhriz

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Tumor Cells ◽

Breast Cancer Cells ◽

Specific Binding ◽

High Specificity ◽

The Novel ◽

Anti Cancer ◽

Therapeutic Peptides ◽

Low Toxicity

: Breast cancer is a heterogeneous malignancy which is the second cause of mortality among women in the world. Increasing the resistance to anti-cancer drugs in breast cancer cells persuades researchers to search the novel therapies approaches for the treatment of the malignancy. Among the novel methods, therapeutic peptides which target and disrupt tumor cells have been of great interest. Therapeutic peptides are short amino acids monomer chains with high specificity to bind and modulate a protein interaction of interest. Several advantages of peptides such as specific binding on tumor cells surface, low molecular weight and low toxicity on normal cells make the peptides as an appealing therapeutic agents against solid tumors, particularly breast cancer. Also, National Institutes of Health (NIH) describes therapeutic peptides as suitable candidate for the treatment of drug-resistant breast cancer. In this review, we attempt to review the different therapeutic peptides against breast cancer cells which can be used in treatment and diagnosis of the malignancy. Meanwhile, we presented an overview of peptide vaccines which have been developed for the treatment of breast cancer.

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750 Malat1 lncRNA controls metastatic reactivation of dormant breast cancer by immune evasion

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0750 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A799-A799

Author(s):

Dhiraj Kumar ◽

Sreeharsha Gurrapu ◽

Hyunho Han ◽

Yan Wang ◽

Seongyeon Bae ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Single Cell ◽

Cancer Cells ◽

Tumor Cells ◽

Immune Evasion ◽

Breast Cancer Cells ◽

Metastatic Potential ◽

Rna Seq

BackgroundLong non-coding RNAs (lncRNAs) are involved in various biological processes and diseases. Malat1 (metastasis-associated lung adenocarcinoma transcript 1), also known as Neat2, is one of the most abundant and highly conserved nuclear lncRNAs. Several studies have shown that the expression of lncRNA Malat1 is associated with metastasis and serving as a predictive marker for various tumor progression. Metastatic relapse often develops years after primary tumor removal as a result of disseminated tumor cells undergoing a period of latency in the target organ.1–4 However, the correlation of tumor intrinsic lncRNA in regulation of tumor dormancy and immune evasion is largely unknown.MethodsUsing an in vivo screening platform for the isolation of genetic entities involved in either dormancy or reactivation of breast cancer tumor cells, we have identified Malat1 as a positive mediator of metastatic reactivation. To functionally uncover the role of Malat1 in metastatic reactivation, we have developed a knock out (KO) model by using paired gRNA CRISPR-Cas9 deletion approach in metastatic breast and other cancer types, including lung, colon and melanoma. As proof of concept we also used inducible knockdown system under in vivo models. To delineate the immune micro-environment, we have used 10X genomics single cell RNA-seq, ChIRP-seq, multi-color flowcytometry, RNA-FISH and immunofluorescence.ResultsOur results reveal that the deletion of Malat1 abrogates the tumorigenic and metastatic potential of these tumors and supports long-term survival without affecting their ploidy, proliferation, and nuclear speckles formation. In contrast, overexpression of Malat1 leads to metastatic reactivation of dormant breast cancer cells. Moreover, the loss of Malat1 in metastatic cells induces dormancy features and inhibits cancer stemness. Our RNA-seq and ChIRP-seq data indicate that Malat1 KO downregulates several immune evasion and stemness associated genes. Strikingly, Malat1 KO cells exhibit metastatic outgrowth when injected in T cells defective mice. Our single-cell RNA-seq cluster analysis and multi-color flow cytometry data show a greater proportion of T cells and reduce Neutrophils infiltration in KO mice which indicate that the immune microenvironment playing an important role in Malat1-dependent immune evasion. Mechanistically, loss of Malat1 is associated with reduced expression of Serpinb6b, which protects the tumor cells from cytotoxic killing by the T cells. Indeed, overexpression of Serpinb6b rescued the metastatic potential of Malat1 KO cells by protecting against cytotoxic T cells.ConclusionsCollectively, our data indicate that targeting this novel cancer-cell-initiated domino effect within the immune system represents a new strategy to inhibit tumor metastatic reactivation.Trial RegistrationN/AEthics ApprovalFor all the animal studies in the present study, the study protocols were approved by the Institutional Animal Care and Use Committee(IACUC) of UT MD Anderson Cancer Center.ConsentN/AReferencesArun G, Diermeier S, Akerman M, et al., Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Genes Dev 2016 Jan 1;30(1):34–51.Filippo G. Giancotti, mechanisms governing metastatic dormancy and reactivation. Cell 2013 Nov 7;155(4):750–764.Gao H, Chakraborty G, Lee-Lim AP, et al., The BMP inhibitor Coco reactivates breast cancer cells at lung metastatic sites. Cell 2012b;150:764–779.Gao H, Chakraborty G, Lee-Lim AP, et al., Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation. Proc Natl Acad Sci U S A 2014 Nov 18; 111(46): 16532–16537.

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Down regulation of Pinkbar/pAKT and MMP2/MMP9 expression in MDA-MB-231 breast cancer cells as potential targets in cancer therapy by hAMSCs secretome

Cells Tissues Organs ◽

10.1159/000520370 ◽

2021 ◽

Author(s):

Termeh Shakery ◽

Fatemeh Safari

Keyword(s):

Breast Cancer ◽

Cancer Therapy ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Molecular Mechanisms ◽

Growth Factor Receptor ◽

3D Cell Culture ◽

Therapeutic Effects ◽

Down Regulation ◽

Egfr Signaling Pathway

Breast cancer (BC) is one of the most causes of cancer-related death among women worldwide. Cancer therapy based on stem cells was considered as a novel and promising platform. In present study, we explored the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through Pinkbar (planar intestinal-and kidney-specific BAR domain protein), pAKT, and matrix metalloproteinases including MMP2, MMP9 on MDA-MB-231 breast cancer cells. To do so, we employed a co-culture system using 6 well plates transwell with a diameter of 0.4 μm pore sized. After 72h hAMSCs-treated MDA-MB-231 breast cancer cells, the expression of Epidermal growth factor receptor (EGFR), and c-Src (a key mediator in EGFR signaling pathway), Pinkbar, pAKT, MMP2, and MMP9 was analyzed by using quantitative real time PCR (qRT-PCR) and western blot methods. Based on using 2D and 3D cell culture models, the significant reduction of tumor cell growth and motility through down regulation of EGFR, c-Src, Pinkbar, pAKT, MMP2, and MMP9 in MDA-MB-231 breast cancer cells was shown. Also, the induction of cellular apoptosis also found. Our finding indicates that the hAMSCS secretome has therapeutic effects on cancer cells. To identify the details of the molecular mechanisms, more experiments will be required.

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Palbociclib and fulvestrant act in synergy to modulate central carbon metabolism in breast cancer cells

10.1101/348722 ◽

2018 ◽

Author(s):

Benedikt Warth ◽

Amelia Palermo ◽

Nicholas J.W. Rattray ◽

Nathan V Lee ◽

Zhou Zhu ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Cancer Cells ◽

Pentose Phosphate Pathway ◽

Metabolic Pathways ◽

Breast Cancer Cells ◽

Pentose Phosphate ◽

List Type ◽

Estrogen Receptor Positive ◽

Positive Breast Cancer

SummaryPalbociclib, is a selective inhibitor of cyclin-dependent kinases 4 and 6 and used as a first-line treatment for patients with estrogen receptor positive breast cancer. It has been shown that patients have improved progression-free survival when treated in combination with fulvestrant, an estrogen receptor antagonist. However, the mechanisms for this survival advantage are not known. We sought to analyze metabolic and transcriptomic changes in MCF-7 adenocarcinoma breast cancer cells following single and combined treatments to determine if selective metabolic pathways are targeted during combination therapy. Our results showed that individually, the drugs caused metabolic disruption to the same metabolic pathways, however fulvestrant additionally attenuated the pentose phosphate pathway and the production of important coenzymes. A comprehensive effect was observed when the drugs were applied together, confirming the combinatory therapy′s synergism in the cell model. This study highlights the power of merging high-dimensional datasets to unravel mechanisms involved in cancer metabolism and therapy.Highlights○First study employing multi-omics to investigate combined therapy on breast cancer cells○Fulvestrant attenuates the pentose phosphate pathway and coenzyme production○Synergism of palbociclib and fulvestrant was confirmed in vitro○Altered key pathways have been identifiedeTOC BlurbJohnson et al. applied an innovative multi-omics approach to decipher metabolic pathways affected by single versus combination dosing of palbociclib and fulvestrant in estrogen receptor positive breast cancer. Key metabolites and genes were correlated within metabolic pathways and shown to be involved in the drugs′ synergism.

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A Nuclear-Directed Ribonuclease Variant Targets Cancer Stem Cells and Inhibits Migration and Invasion of Breast Cancer Cells

Cancers ◽

10.3390/cancers13174350 ◽

2021 ◽

Vol 13 (17) ◽

pp. 4350

Author(s):

Jessica Castro ◽

Giusy Tornillo ◽

Gerardo Ceada ◽

Beatriz Ramos-Neble ◽

Marlon Bravo ◽

...

Keyword(s):

Breast Cancer ◽

Stem Cells ◽

Cancer Stem Cells ◽

Tumor Cell ◽

Cancer Cells ◽

Tumor Cells ◽

Cell Lines ◽

Breast Cancer Cells ◽

Migration And Invasion ◽

Tumor Cell Lines

Despite the significant advances in cancer research made in recent years, this disease remains one of the leading causes of death worldwide. In part, this is due to the fact that after therapy, a subpopulation of self-renewing tumor cells can survive and promote cancer relapse, resistance to therapies and metastasis. Targeting these cancer stem cells (CSCs) is therefore essential to improve the clinical outcome of cancer patients. In this sense, multi-targeted drugs may be promising agents targeting CSC-associated multifocal effects. We have previously constructed different human pancreatic ribonuclease (RNase) variants that are cytotoxic for tumor cells due to a non-classical nuclear localization signal introduced in their sequence. These cytotoxic RNases affect the expression of multiple genes involved in deregulated metabolic and signaling pathways in cancer cells and are highly cytotoxic for multidrug-resistant tumor cell lines. Here, we show that these cytotoxic nuclear-directed RNases are highly selective for tumor cell lines grown in 3D, inhibit CSCs’ development and diminish the self-renewal capacity of the CSCs population. Moreover, these human RNase variants reduce the migration and invasiveness of highly invasive breast cancer cells and downregulate N-cadherin expression.

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Calcineurin regulates the stability and activity of estrogen receptor α

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114258118 ◽

2021 ◽

Vol 118 (44) ◽

pp. e2114258118

Author(s):

Takahiro Masaki ◽

Makoto Habara ◽

Yuki Sato ◽

Takahiro Goshima ◽

Keisuke Maeda ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Cancer Cells ◽

Cancer Progression ◽

Breast Cancer Cells ◽

Molecular Mechanisms ◽

Target Genes ◽

Estrogen Receptor Α ◽

The Stability ◽

Positive Breast Cancer

Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser294 in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser294 promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser118 by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to β-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser294 and Ser118. Finally, the expression of the calcineurin A–α gene (PPP3CA) was associated with poor prognosis in ER-α–positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α–positive breast cancer.

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ZFHX3 Promotes the Proliferation and Tumor Growth of ER-Positive Breast Cancer Cells Likely by Enhancing Stem-Like Features and MYC and TBX3 Transcription

Cancers ◽

10.3390/cancers12113415 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3415

Author(s):

Ge Dong ◽

Gui Ma ◽

Rui Wu ◽

Jinming Liu ◽

Mingcheng Liu ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Growth ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Molecular Mechanisms ◽

Mammary Epithelial Cells ◽

Progesterone Receptors ◽

Mammary Epithelial ◽

Luciferase Reporter ◽

Mammosphere Formation

Breast cancer is a common malignancy, but the understanding of its cellular and molecular mechanisms is limited. ZFHX3, a transcription factor with many homeodomains and zinc fingers, suppresses prostatic carcinogenesis but promotes tumor growth of liver cancer cells. ZFHX3 regulates mammary epithelial cells’ proliferation and differentiation by interacting with estrogen and progesterone receptors, potent breast cancer regulators. However, whether ZFHX3 plays a role in breast carcinogenesis is unknown. Here, we found that ZFHX3 promoted the proliferation and tumor growth of breast cancer cells in culture and nude mice; and higher expression of ZFHX3 in human breast cancer specimens was associated with poorer prognosis. The knockdown of ZFHX3 in ZFHX3-high MCF-7 cells decreased, and ZFHX3 overexpression in ZFHX3-low T-47D cells increased the proportion of breast cancer stem cells (BCSCs) defined by mammosphere formation and the expression of CD44, CD24, and/or aldehyde dehydrogenase 1. Among several transcription factors that have been implicated in BCSCs, MYC and TBX3 were transcriptionally activated by ZFHX3 via promoter binding, as demonstrated by luciferase-reporter and ChIP assays. These findings suggest that ZFHX3 promotes breast cancer cells’ proliferation and tumor growth likely by enhancing BCSC features and upregulating MYC, TBX3, and others.

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A Novel Human Phosphatidylethanolamine-binding Protein Resists Tumor Necrosis Factor α-induced Apoptosis by Inhibiting Mitogen-activated Protein Kinase Pathway Activation and Phosphatidylethanolamine Externalization

Journal of Biological Chemistry ◽

10.1074/jbc.m405147200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45855-45864 ◽

Cited By ~ 75

Author(s):

Xiaojian Wang ◽

Nan Li ◽

Bin Liu ◽

Hongying Sun ◽

Taoyong Chen ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Necrosis Factor ◽

Cancer Cells ◽

Tumor Cells ◽

Breast Cancer Cells ◽

Tumor Necrosis ◽

Induced Apoptosis ◽

Phosphatidylethanolamine Binding Protein ◽

Necrosis Factor ◽

Mcf 7

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) α treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFα stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFα-induced ERK1/2, MEK1, and JNK activation and TNFα-mediated apoptosis. Co-precipitation andin vitroprotein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFα. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFα-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.

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TOPOGRAPHY MEDIATED REGULATION OF HER-2 EXPRESSION IN BREAST CANCER CELLS

Nano LIFE ◽

10.1142/s1793984412410097 ◽

2012 ◽

Vol 02 (03) ◽

pp. 1241009 ◽

Cited By ~ 5

Author(s):

AMITA DAVEREY ◽

AUSTIN C. MYTTY ◽

SRIVATSAN KIDAMBI

Keyword(s):

Breast Cancer ◽

Cell Adhesion ◽

Cancer Cells ◽

Cancer Progression ◽

Breast Tumor ◽

Cancer Biology ◽

Breast Cancer Cells ◽

Molecular Mechanisms ◽

Her 2 ◽

Micro Topography

This article demonstrates that the surface micro-topography regulates the biology of breast cancer cells, including the expression of HER-2 gene and protein. The breast tumor microenvironment is made up of heterogenous mixture of pores, ridges and collagen fibers with well defined topographical features. Although, significant progress has been achieved towards elucidating the biochemical and molecular mechanisms that underlie breast cancer progression, quantitative characterization of the associated mechanical/topographical properties and their role in breast tumor progression remains largely unexplored. Therefore, the aim of this study is to investigate the effect of topography on the adhesion and biology of breast cancer cells in in vitro cultures. Polydimethylsiloxane (PDMS) surfaces containing different topographies were coated with polyelectrolyte multilayers (PEMs) to improve cell adhesion and maintain cell culture. HER-2 expressing breast cancer cells, BT-474 and SKBr3, were cultured on these PDMS surfaces. We demonstrate that micro-topography affects the cell adhesion and distribution depending on the topography on the PDMS surfaces. We also report for the first time that surface topography down-regulates the HER-2 gene transcription and protein expression in breast cancer cells when cultured on PDMS surfaces with micro-topographies compared to the tissue culture polystyrene surface (TCPS) control. Results from this study indicate that micro-topography modulates morphology of cells, their distribution and expression of HER-2 gene and protein in breast cancer cells. This study provides a novel platform for studying the role of native topography in the progression of breast cancer and has immense potential for understanding the breast cancer biology.

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