PI3-K/Akt-mediated anoikis resistance of human osteosarcoma cells requires Src activation

doi:10.1016/j.ejca.2006.03.007

European Journal of Cancer

Volume 42, Issue 10, July 2006, Pages 1491-1500

https://doi.org/10.1016/j.ejca.2006.03.007 Get rights and content

Abstract

Considerable advances in understanding the mechanisms associated with anoikis resistance of normal and malignant epithelial cells have been made. However, little is still known about the pathways involved in anoikis resistance of non-epithelial cells such as fibroblasts and sarcomas. Our results show that Src activity contributes to anoikis resistance of human osteosarcoma SAOS-2 cells. Src was found to be upregulated in anoikis resistant SAOS cells, and pharmacological inhibition of its activity resulted in the restoration of anoikis sensitivity. A normal pattern of dephosphorylation of FAK was observed upon cell detachment of both anoikis sensitive and resistant SAOS-2 cells, suggesting that FAK activity during anoikis resistance is not essential. The activity of Akt was found to be upregulated in anoikis resistant SAOSar cells and the pharmacological inhibition of PI3-K activity restored sensitivity to anoikis resistant cells, reconfirming the critical role of PI3-K/Akt pathway in cell survival. Furthermore, pharmacological inhibition of Src resulted in a decrease of Akt phosphorylation at Ser473. Altogether, these studies indicated a survival pathway mediated by the Src-dependent activation of the PI3-K/Akt pathway in a manner independent of FAK activity.

Introduction

Anoikis, Greek for “homelessness”, is a form of apoptosis triggered by the lack of cell survival signals generated from the interactions with the ECM.1, 2 Physiological anoikis is considered to be important in the maintenance of homeostasis and tissue architecture.³ Although described initially in epithelial and endothelial cells, anoikis has now also been found in normal cells of non-epithelial origin such as fibroblasts, hepatocytes, and pancreatic islets.4, 5, 6, 7, 8, 9 Furthermore, we and others have shown that malignant non-epithelial cells are sensitive to anoikis as well.10, 11, 12

The importance of anoikis in maintaining normal cell turnover and tissue architecture is evident however, the role of cellular resistance to anoikis is not as clear. Cells that are resistant to anoikis would live longer unattached and therefore have roles in cell migration and tissue remodelling. On the other hand, aberrant regulation of anoikis resulting in pathologic anoikis resistance has been described as one of the hallmarks of malignant transformation.¹³ Anoikis resistance affords transformed cells increased survival times in the absence of matrix attachment, facilitating their migration and colonization of secondary sites.14, 15, 16, 17 We have shown that acquisition of anoikis resistance also applies to non-epithelial malignancies such as osteosarcoma. In our studies, cyclic manipulation of adhesion receptors in human osteosarcoma can convert anoikis sensitive cells to a stable and heritable anoikis resistant phenotype.¹⁸

Several mechanisms associated with anoikis resistance of cells from epithelial origin have been described recently.¹⁹ However, very little is known about the pathways associated with anoikis resistance of cells of non-epithelial origin, such as fibroblasts or sarcomas. Understanding the mechanisms and role of anoikis resistance during the progression and metastasis of osteosarcoma is of clinical importance since 30% to 40% of patients develop lung metastasis despite aggressive chemotherapy and surgical resection of the primary lesion.20, 21

It has been generally accepted that survival of adhered cells is maintained by signals generated by interactions between integrins and the ECM. Such signals are in turn transmitted to the cytoplasm by components of the focal adhesions, in which FAK is the central player. The major site of FAK autophosphorylation, Tyr397, is vital for the biological and biochemical functions of FAK, and represents the docking site for SH2-containing signalling molecules such as Src, Shc and the p85 subunit of PI3-K.²² Ultimately, cell survival is associated mostly with the downstream activation of the PI3-K/Akt pathway, and since Akt activity depends heavily on the availability of PIP3, phosphatases such as PTEN and SHIP ²³ act as potent negative regulators of its activity.

Direct involvement of FAK and Src during adhesion-mediated cell survival has been documented.²⁴ Expression of activated FAK results in the acquisition of anoikis resistance in MDCK cells, and conversely, inhibition of FAK function results in cell detachment and apoptosis.25, 26, 27 Moreover, the initial observation that v-Src transformed epithelial cells became anoikis resistant suggested a role for Src in adhesion-mediated survival as well.¹ This has been corroborated by studies in colon and breast carcinomas in which expression of activated Src leads to increased anoikis resistance.²⁸ Furthermore, a recent report has shown that in breast carcinoma cells Src acts downstream of FAK in preventing anoikis.²⁹ Src-mediated anoikis resistance in a FAK activation dependent manner has also been described in normal breast and gallbladder epithelial cells.³⁰ However, recent reports have shown that Src-mediated anoikis resistance can occur independently of FAK activation.³¹ Thus, this questions the universal importance of FAK in anchorage-independent cell survival, whether different cell types use different pathways, and whether the preservation of such pathways persists during embryonic development or malignant transformation.

Therefore the purpose of our study was to identify the key mediators of anoikis resistance in human osteosarcoma cells. Our results suggest a survival pathway mediated by the Src-dependent activation of the PI3-K/Akt pathway in a manner independent of FAK activity.

Section snippets

Cell culture and reagents

The parental human osteosarcoma cell line SAOS-2 (SAOSp) was obtained from the American Type Culture Collection (Manassas, VA). SAOSp cells were maintained in Eagle’s MEM (BioWhittaker, Walkersville, MD), supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MD), 2 mM L-glutamine, 1 mM sodium pyruvate and non-essential amino acids (Sigma, St. Louis, MO). Anoikis resistant SAOS cells (SAOSar) were generated by sequential cycles of culture on untreated (adhered) and poly-HEMA

Resistance to anoikis of SAOSar cells involves the activation of Src

SAOSp cells undergo anoikis when proper adherence to the ECM is denied.¹¹ Nevertheless, an anoikis resistant subline, SAOSar, has been developed by alternating cycles of culture under suspended and adhered conditions.¹⁸ Apoptosis was determined by PI staining followed by flow cytometry analyses of DNA content. The percentage of cells in the sub-G/0 phase is representative of apoptosis. Fig. 1A shows that when both SAOSp and SAOSar cells are allowed to adhere to cell culture treated wells very

Discussion

Our study focused on the mediators associated with anoikis resistance of human osteosarcoma, SAOS-2 cells. We found the activity of Src necessary for survival under suspended conditions. Since v-Src transformation results in anchorage independent growth, for which anoikis resistance would be a pre-requisite, our findings are consistent for a role of c-Src in adhesion independent survival.

Src activation in response to engagement of integrins with ECM components has been reported.²⁴ However,

Conflict of interest statement

None declared.

Acknowledgements

The authors would like to thank Ricky Rojas for technical support and Karen Ramirez for flow cytometry analyses. This study was supported by the NIH grants T32DE015355-01, CA 62596 and CA 166672 Cancer Center Support Core Grant, a Kleberg Fund for Innovative Research Institutional Grant and by the Onstead Foundation for Osteosarcoma Research.

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PI3-K/Akt-mediated anoikis resistance of human osteosarcoma cells requires Src activation

Abstract

Introduction

Section snippets

Cell culture and reagents

Resistance to anoikis of SAOSar cells involves the activation of Src

Discussion

Conflict of interest statement

Acknowledgements

Bone

Mol Genet Metab

Surgery

Neuroscience

Cell

Eur J Cancer

Curr Opin Cell Biol

Prog Biophys Mol Biol

Cell Biol Int

J Biol Chem

Anal Biochem

Cell

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

Curr Opin Cell Biol

Cell

Disruption of epithelial cell-matrix interactions induces apoptosis

J Cell Biol

The extracellular matrix as a cell survival factor

Mol Biol Cell

Anoikis in the cardiovascular system: known and unknown extracellular mediators

Arterioscler Thromb Vasc Biol

Merosin-integrin promotion of skeletal myofiber cell survival: Differentiation state-distinct involvement of p60Fyn tyrosine kinase and p38alpha stress-activated MAP kinase

J Cell Physiol

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumour cell survival

Cancer Res

Insulin-like growth factor-I-mediated survival from anoikis: role of cell aggregation and focal adhesion kinase

J Cell Physiol

Alpha 4 integrin increases anoikis of human osteosarcoma cells

J Cell Biochem

Anoikis and metastatic potential of cloudman S91 melanoma cells

Cancer Res

Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB

Nature

Apoptosis, anoikis and their relevance to the pathobiology of colon cancer

Pathol Int