Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion

Joshi, Bharat; Strugnell, Scott S.; Goetz, Jacky G.; Kojic, Liliana D.; Cox, Michael; Griffith, Obi L.; Chan, Simon K.; Jones, Steven J.M.; Leung, Sher-Ping; Masoudi, Hamid; Leung, Samuel; Wiseman, Sam M.; Nabi, Ivan R.

doi:10.1158/0008-5472.can-08-0343

Cited by 240 publications

(285 citation statements)

References 48 publications

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“…Further, c-Src-mediated caveolin-1 phosphorylation is reported to stimulate the subcellular trafficking of activated EGF-R augmenting cell migration and anchorage-independent growth [57][58][59]. To this end, Joshi et al [60] have recently shown phosphorylated caveolin-1 to co-operate with Rho/ROCK and FAK-dependent signalling pathways promoting tumour cell invasion and metastasis in breast cancer. Such studies are consistent with clinical data showing a positive correlation between caveolin-1 expression (although distinction between phosphorylated and nonphosphorylated forms not addressed) and poor patient survival in highly aggressive invasive breast cancers [42][43][44]61].…”

Section: Discussionmentioning

confidence: 99%

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Thomas

Hutcheson

Campbell

et al. 2009

Breast Cancer Res Treat

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Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance. Breast Cancer Research and Treatment, Springer Verlag, 2009, 119 (3) Abstract Caveolin-1 displays both tumour-suppressor and tumour-promoter properties in breast cancer. Using characterised preclinical cell models for the transition of oestrogen-sensitive (WT-MCF-7 cells) to a tamoxifenresistant (TAM-R cells) phenotype we examined the role caveolin-1 in the development of hormone-resistant breast cancer. The WT-MCF-7 cells showed abundant expression of caveolin-1 which potentiated oestrogen-receptor (ERa) signalling and promoted cell growth despite caveolin-1 mediating inhibition of ERK signalling. In TAM-R cells caveolin-1 expression was negligible, repressed by EGF-R/ERK signalling. Pharmacological inhibition of EGFR/ERK in TAM-R cells restored caveolin-1 and also resulted in the emergence of pools of phosphorylated caveolin-1. WT-MCF-7 cells exposed to tamoxifen for upto 12 weeks displayed increased caveolin-1 (peaking by week 2) followed (after week 8) by a marked decrease as the cells progress to develop a stable tamoxifen-resistant phenotype. The targeted down-regulation (siRNA) of caveolin-1 in WT-MCF-7 cells reduced growth but did not affect their sensitivity to tamoxifen, suggesting loss of caveolin-1 alone is not sufficient to confer tamoxifen-resistance. Hyperactivation of EGFR/ERK is a feature of tamoxifen-resistant breast cancer cells, a principal driver of cell growth. Recombinant expression of caveolin-1 in TAM-R cells did not affect EGFR/ERK activity, potentially due to mislocalisation of caveolin-1 through hyperactivation of the mTOR pathway or altered caveolin-1 phosphorylation. This work defines a novel role for caveolin-1 with implications for the clinical course of breast cancer and identifies caveolin-1 as a potential drug target for the treatment of early oestrogen-dependent breast cancers. Further, the loss of caveolin-1 may have benefit as a molecular signature for tamoxifen resistance.

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Section: Discussionmentioning

confidence: 99%

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Thomas

Hutcheson

Campbell

et al. 2009

Breast Cancer Res Treat

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“…Control and KAI1-specific siRNA smart pools were transiently transfected in to MCF7 or HEK293 stable cell lines using Lipofectamine 2000 (Invitrogen) following the protocol described before (23). KAI1 knockdown was assessed by Western blotting.…”

Section: Methodsmentioning

confidence: 99%

“…MTT Assay-Cell proliferation assays were performed as described previously (23). Briefly, 10,000 cells were seeded on 96-well plates for 48 -72 h, prior to adding MTT reagent diluted 1:10 from 5 mg/ml stock in RPMI (MCF7; MDA-435) or Dulbecco's modified Eagle's medium (HEK293) medium, after which the cells were allowed to grow in a CO 2 incubator in the dark for 4 -5 h. The medium was decanted, precipitates of metabolite (Formazan) were dissolved in 200 l of Me 2 SO, and absorbance was measured at 570 nm.…”

Section: Volume 285 • Number 12 • March 19 2010mentioning

confidence: 99%

A Role for KAI1 in Promotion of Cell Proliferation and Mammary Gland Hyperplasia by the gp78 Ubiquitin Ligase

Joshi

Nabi

2010

Journal of Biological Chemistry

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Expression of gp78, an E3 ubiquitin ligase in endoplasmic reticulum-associated degradation, is associated with tumor malignancy. To study gp78 overexpression in mammary gland development and tumorigenicity, we generated murine mammary tumor virus (MMTV) long terminal repeat-driven gp78 transgenic mice. Embryos carrying the gp78 transgene cassette were implanted in FVB surrogate mothers, and two founders with high copy integration showed elevated gp78 expression at both transcript and protein levels at the virgin stage and at 12 days gestation. Transgenic mammary glands showed increased ductal branching, dense alveolar lobule formation, and secondary terminal end bud development. Bromodeoxyuridine staining showed increased proliferation in hyperplastic ductal regions at the virgin stage and at 12 days gestation compared with wild type mice. Reduced expression of the metastasis suppressor KAI1, a gp78 endoplasmic reticulum-associated degradation substrate, demonstrates that gp78 ubiquitin ligase activity is increased in MMTV-gp78 mammary gland. Similarly, metastatic MDA-435 cells exhibit increased gp78 expression, decreased KAI1 expression, and elevated proliferation compared with nonmetastatic MCF7 cells whose proliferation was enhanced upon knockdown of KAI1. Importantly, stable gp78 knockdown HEK293 cells showed increased KAI1 expression and reduced proliferation that was rescued upon KAI1 knockdown, demonstrating that gp78 regulation of cell proliferation is mediated by KAI1. Mammary tumorigenesis was not observed in repeatedly pregnant MMTV-long terminal repeat-gp78 transgenic mice over a period of 18 months post-birth. Elevated gp78 ubiquitin ligase activity is therefore not sufficient for mammary tumorigenesis. However, the hyperplastic phenotype observed in mammary glands of MMTV-gp78 transgenic mice identifies a novel role for gp78 expression in enhancing mammary epithelial cell proliferation and nontumorigenic ductal outgrowth.gp78, also called autocrine motility factor receptor, is an E3 ubiquitin ligase implicated in endoplasmic reticulum-associated degradation that also has a dual function as a cell surface cytokine receptor for autocrine motility factor/phosphoglucose (1). gp78 overexpression is closely linked to tumor malignancy and human cancer (2) and was identified as one of the 189 most mutated genes in breast and colon cancers (3). gp78 expression correlates with aggressive tumor biology and poor outcome for malignancies of the lung, tongue, esophagus, stomach, colon, rectum, liver, breast, thymus, and skin (2). Notably, in bladder, colorectal, gastric, skin, and esophageal cancers (4 -8), gp78 is either not expressed or expressed at significantly reduced levels in adjacent normal tissue. However, whereas overexpression of gp78 in tumors is well documented, it has yet to be determined whether gp78 overexpression contributes to tumor progression.Substrates of gp78 E3 ubiquitin ligase activity include CD3-␦, the T cell receptor, apoB lipoprotein, hydroxymethylglutarylCoA reductase, cystic fibrosi...

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“…These results indicate that arachidonic acid-induced adhesion of MDA-MB-435 cells to type IV collagen requires both Rho activation and ROCK II signaling. Several proteins have been implicated as effectors of ROCK signaling, including myosin light chain (50), cofilin (51), and caveolin (52). We examined each of these in MDA-MB-435 cells and did not identify any ROCK-dependent changes in phosphorylation after treatment with arachidonic acid (data not shown), indicating that although Rho/ROCK is critical for the fatty acid-induced adhesion, the signaling apparently functions through other downstream effectors.…”

Section: Rock Is Necessary For Adhesion Of Mda-mb-435 Cells To Collagmentioning

confidence: 98%

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Garcia

Ray

Lackford

et al. 2009

Journal of Biological Chemistry

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Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogenactivated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.The ability of tumor cells to metastasize to secondary sites is a hallmark of neoplastic disease. Unfortunately, this propensity to spread is the primary cause of morbidity and death in cancer patients (1). Metastasis is clearly a highly regulated, multistep process that occurs in a spatiotemporal manner (2-4). To escape the restrictive compartment boundaries characteristic of adult tissue, separate intravasation and extravasation steps requiring alterations in co-adhesion, adhesion, invasion, and migration must occur. Execution of these biological processes, involving multiple proteins and cellular organelles, require highly coordinated cell signaling mechanisms.The Rho family of small GTPases regulates many facets of cytoskeletal rearrangements that facilitate cell attachment and migration (5-7). Rho GTPases act as molecular switches by changing from an inactive GDP-bound conformation to an active GTPbound conformation, thereby regulating a signaling pathway. These proteins are directly regulated by Rho guanine nucleotide exchange factors (GEFs), 2 Rho GTPase activating proteins, and Rho GDP-dissociation inhibitors (8 -12). RhoGEFs bind to the GTPase to catalyze the dissociation of GDP, allowing the binding of GTP and thereby promoting Rho activation (8). The RGS (regulators of G protein signaling) domain-c...

show abstract

Phosphorylated Caveolin-1 Regulates Rho/ROCK-Dependent Focal Adhesion Dynamics and Tumor Cell Migration and Invasion

Cited by 240 publications

References 48 publications

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

A Role for KAI1 in Promotion of Cell Proliferation and Mammary Gland Hyperplasia by the gp78 Ubiquitin Ligase

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

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