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HER2-mediated GLI2 stabilization promotes anoikis resistance and metastasis of breast cancer cells.

ABSTRACT: Breast cancer metastasis is a multi-step process and requires cells to overcome anoikis. Anoikis is defined as cell-death that occurs due to loss of cell adhesion. During the course of cancer progression, tumor cells acquire resistance to anoikis. However, mechanisms of anoikis resistance are not clear. Human epidermal growth receptor 2 (HER2) overexpressing breast tumors are known to be highly aggressive and metastatic. The mechanisms correlating HER2 with metastasis are poorly understood. We observed increased anoikis resistance in HER2 overexpressing breast cancer cells. In addition, we identified that HER2 overexpression was also associated with increased sonic hedgehog (SHH) signaling especially GLI2, and that inhibition of SHH pathway suppressed anoikis resistance. GSK3? is known to facilitate proteasome-mediated degradation of GLI2. Moreover, we observed that silencing of GLI2 resulted in reduced migration and invasion of HER2 overexpressing cells. Anoikis resistant HER2 overexpressing cells also showed increased rate and extent of metastasis in vivo, as compared to wild type anoikis resistant cells. Taken together, this study indicates a novel role of HER2/GSK3?/GLI2 axis in anoikis resistance and metastasis, and that GLI2 could be a potential target for anti-cancer therapies.

SUBMITTER: Gupta P

PROVIDER: S-EPMC6311434 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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HER2-mediated GLI2 stabilization promotes anoikis resistance and metastasis of breast cancer cells.

Gupta Parul P Gupta Nehal N Fofaria Neel M NM Ranjan Alok A Srivastava Sanjay K SK

Cancer letters 20181026

Breast cancer metastasis is a multi-step process and requires cells to overcome anoikis. Anoikis is defined as cell-death that occurs due to loss of cell adhesion. During the course of cancer progression, tumor cells acquire resistance to anoikis. However, mechanisms of anoikis resistance are not clear. Human epidermal growth receptor 2 (HER2) overexpressing breast tumors are known to be highly aggressive and metastatic. The mechanisms correlating HER2 with metastasis are poorly understood. We o ...[more]

PMID: 30409762

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Project description:BACKGROUND:Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis. METHODS:We analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with ?1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student's t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study. RESULTS:Collagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced ?1 integrin activation. Blocking ?1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis. CONCLUSIONS:Our results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.

Project description:BackgroundEpstein-Barr virus (EBV) is the first discovered human tumor virus that is associated with a variety of malignancies of both lymphoid and epithelial origin including nasopharyngeal carcinoma (NPC). The EBV-encoded latent membrane protein 1 (LMP1) has been well-defined as a potent oncogenic protein, which is intimately correlated with NPC pathogenesis. Anoikis is considered to be a physiological barrier to metastasis, and avoiding anoikis is a major hallmark of metastasis. However, the role of LMP1 in anoikis-resistance and metastasis of NPC has not been fully identified.MethodsTrypan blue staining, colony formation assay, flow cytometry, and TUNEL staining, as well as the detection of apoptosis and anoikis resistance-related markers was applied to evaluate the anoikis-resistant capability of NPC cells cultured in ultra-low adhesion condition. Co-immunoprecipitation (Co-IP) experiment was performed to determine the interaction among LMP1, PRMT1 and PGC-1α. Ex vivo ubiquitination assay was used to detect the ubiquitination level of PGC-1α. Anoikis- resistant LMP1-positive NPC cell lines were established and applied for the xenograft and metastatic animal experiments.ResultsOur current findings reveal the role of LMP1-stabilized peroxisome proliferator activated receptor coactivator-1a (PGC-1α) in anoikis resistance and immune escape to support the invasion and metastasis of NPC. Mechanistically, LMP1 enhances PGC-1α protein stability by promoting the interaction between arginine methyltransferase 1 (PRMT1) and PGC-1α to elevate the methylation modification of PGC-1α, thus endowing NPC cells with anoikis-resistance. Meanwhile, PGC-1α mediates the immune escape induced by LMP1 by coactivating with STAT3 to transcriptionally up-regulate PD-L1 expression.ConclusionOur work provides insights into how virus-encoded proteins recruit and interact with host regulatory elements to facilitate the malignant progression of NPC. Therefore, targeting PGC-1α or PRMT1-PGC-1α interaction might be exploited for therapeutic gain for EBV-associated malignancies.

Project description:BackgroundThe transforming growth factor-beta (TGF-beta) pathway, which has both tumor suppressor and pro-oncogenic activities, is often constitutively active in melanoma and is a marker of poor prognosis. Recently, we identified GLI2, a mediator of the hedgehog pathway, as a transcriptional target of TGF-beta signaling.MethodsWe used real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting to determine GLI2 expression in human melanoma cell lines and subsequently classified them as GLI2high or as GLI2low according to their relative GLI2 mRNA and protein expression levels. GLI2 expression was reduced in a GLI2high cell line with lentiviral expression of short hairpin RNA targeting GLI2. We assessed the role of GLI2 in melanoma cell invasiveness in Matrigel assays. We measured secretion of matrix metalloproteinase (MMP)-2 and MMP-9 by gelatin zymography and expression of E-cadherin by western blotting and RT-PCR. The role of GLI2 in development of bone metastases was determined following intracardiac injection of melanoma cells in immunocompromised mice (n = 5-13). Human melanoma samples (n = 79) at various stages of disease progression were analyzed for GLI2 and E-cadherin expression by immunohistochemistry, in situ hybridization, or RT-PCR. All statistical tests were two-sided.ResultsAmong melanoma cell lines, increased GLI2 expression was associated with loss of E-cadherin expression and with increased capacity to invade Matrigel and to form bone metastases in mice (mean osteolytic tumor area: GLI2high vs GLI2low, 2.81 vs 0.93 mm(2), difference = 1.88 mm(2), 95% confidence interval [CI] = 1.16 to 2.60, P < .001). Reduction of GLI2 expression in melanoma cells that had expressed high levels of GLI2 substantially inhibited both basal and TGF-beta-induced cell migration, invasion (mean number of Matrigel invading cells: shGLI2 vs shCtrl (control), 52.6 vs 100, difference = 47.4, 95% CI = 37.0 to 57.8, P = .024; for shGLI2 + TGF-beta vs shCtrl + TGF-beta, 31.0 vs 161.9, difference = -130.9, 95% CI = -96.2 to -165.5, P = .002), and MMP secretion in vitro and the development of experimental bone metastases in mice. Within human melanoma lesions, GLI2 expression was heterogeneous, associated with tumor regions in which E-cadherin was lost and increased in the most aggressive tumors.ConclusionGLI2 was directly involved in driving melanoma invasion and metastasis in this preclinical study.

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HER2-mediated GLI2 stabilization promotes anoikis resistance and metastasis of breast cancer cells.

Publications

HER2-mediated GLI2 stabilization promotes anoikis resistance and metastasis of breast cancer cells.

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