Unlike normal cells, transformed cells are unable to grow when methionine in the growth media is restricted. Reversion to methionine independence is a rare event in transformed and malignant cells. ...Methionine-independent revertants provide an excellent system to identify metabolic signatures and molecular characteristics associated with methionine dependency of transformed cells. Revertants maintain the genetic background and general growth behavior of the parental cell line, except that they proliferate under methionine restriction such as in methionine-free media supplemented with homocysteine. Here we describe a general approach to generate methionine-independent revertants using the example of the triple-negative breast cancer cell line MDA-MB-468. To validate and characterize reversion we describe assays to evaluate cell proliferation and anchorage-independent growth in soft agar.
In cancer, two unique and seemingly contradictory behaviors are evident: on the one hand, tumors are typically stiffer than the tissues in which they grow, and this high stiffness promotes their ...malignant progression; on the other hand, cancer cells are anchorage-independent-namely, they can survive and grow in soft environments that do not support cell attachment. How can these two features be consolidated? Recent findings on the mechanisms by which cells test the mechanical properties of their environment provide insight into the role of aberrant mechanosensing in cancer progression. In this review article, we focus on the role of high stiffness on cancer progression, with particular emphasis on tumor growth; we discuss the mechanisms of mechanosensing and mechanotransduction, and their dysregulation in cancerous cells; and we propose that a 'yin and yang' type phenomenon exists in the mechanobiology of cancer, whereby a switch in the type of interaction with the extracellular matrix dictates the outcome of the cancer cells.
Integrin dependent regulation of growth factor signalling confers anchorage dependence that is deregulated in cancers. Downstream of integrins and oncogenic Ras the small GTPase Ral is a vital ...mediator of adhesion dependent trafficking and signalling. This study identifies a novel regulatory crosstalk between Ral and Arf6 that controls Ral function in cells. In re-adherent mouse fibroblasts (MEFs) integrin dependent activation of RalA drives Arf6 activation. Independent of adhesion constitutively active RalA and RalB could both however activate Arf6. This is further conserved in oncogenic H-Ras containing bladder cancer T24 cells, which express anchorage independent active Ral that supports Arf6 activation. Arf6 mediates active Ral-exocyst dependent delivery of raft microdomains to the plasma membrane that supports anchorage independent growth signalling. Accordingly in T24 cells the RalB-Arf6 crosstalk is seen to preferentially regulate anchorage independent Erk signalling. Active Ral we further find uses a Ral-RalBP1-ARNO-Arf6 pathway to mediate Arf6 activation. This study hence identifies Arf6, through this regulatory crosstalk, to be a key downstream mediator of Ral isoform function along adhesion dependent pathways in normal and cancer cells.
•Ral mediates Arf6 activation downstream of integrins and oncogenic Ras.•Arf6 mediates Ral-exocyst dependent delivery of raft microdomains.•Active Ral supports anchorage independent Arf6 activation in bladder cancer T24 cells.•Ral-Arf6 crosstalk in T24 cells regulates anchorage independent Erk signalling.•A Ral-RalBP1-ARNO-Arf6 pathway mediates the Ral-Arf6 crosstalk.
During all stages of tumor progression, cancer cells are subjected to inappropriate extracellular matrix environments and must undergo adaptive changes in order to evade growth constraints associated ...with the loss of matrix attachment. A gain of function screen for genes that enable proliferation independently of matrix anchorage identified a cell adhesion molecule PVRL4 (poliovirus-receptor-like 4), also known as Nectin-4. PVRL4 promotes anchorage-independence by driving cell-to-cell attachment and matrix-independent integrin β4/SHP-2/c-Src activation. Solid tumors frequently have copy number gains of the PVRL4 locus and some have focal amplifications. We demonstrate that the transformation of breast cancer cells is dependent on PVRL4. Furthermore, growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer. DOI:http://dx.doi.org/10.7554/eLife.00358.001.
MDR1 expression identifies human melanoma stem cells Keshet, Gilmor I.; Goldstein, Itamar; Itzhaki, Orit ...
Biochemical and biophysical research communications,
04/2008, Volume:
368, Issue:
4
Journal Article
Peer reviewed
ABC transporters are often found to be inherently expressed in a wide variety of stem cells, where they provide improved protection from toxins. We found a subpopulation of human melanoma cells ...expressing multidrug-resistance gene product 1 (MDR1). This fraction co-expresses the ABC transporters, ABCB5 and ABCC2 in addition to the stem cell markers, nanog and human telomerase reverse transcriptase (hTERT). The clonogenicity and self-renewal capacity of MDR1
+ melanoma cells were investigated in single cell settings using the limiting dilution assay. We found that the MDR1
+ cells, isolated by FACS sorting, demonstrated a higher self-renewal capacity than the MDR1
− fraction, a key stem cell feature. Moreover, MDR1
+ cells had higher ability to form spheres in low attachment conditions, a hallmark of cancer. In conclusion, these novel findings imply that the MDR1
+ cells represent melanoma stem cells and thus should be considered as a unique cellular target for future anti-melanoma therapies.
Abstract Metastasis formation is an essential aspect of cancer, for which the molecular underpinning has long been subject to debate. Although the organ preference for dissemination is governed by ...tumor–host interactions on the epigenetic level there is a genetic basis to the ability of cancer cells to disseminate. Metastasis genes encode homing receptors, their ligands, and extracellular matrix-degrading proteinases, which jointly cause invasion and anchorage-independence. They are developmentally non-essential stress response genes that physiologically mediate the homing of immune system cells. Metastatic potential is conferred to cancer cells by aberrant expression or splicing of these genes. Oncogenes act upstream of metastasis genes. In cancer cells, oncogenic signaling activates distinct genetic programs leading to cell cycle progression and invasiveness, respectively. The expression of metastasis genes is regulated by multi-subunit transcription factor complexes. The identification of genes that direct cancer metastasis implicates them as candidate drug targets.
Adaptation and survival of cancer cells to various stress and growth factor conditions is crucial for successful metastasis. A double-negative feedback loop between two serine/threonine kinases AMPK ...(AMP-activated protein kinase) and Akt can regulate the adaptation of breast cancer cells to matrix-deprivation stress. This feedback loop can significantly generate two phenotypes or cell states: matrix detachment-triggered pAMPK
/ pAkt
state, and matrix (re)attachment-triggered pAkt
/ pAMPK
state. However, whether these two cell states can exhibit phenotypic plasticity and heterogeneity in a given cell population, i.e., whether they can co-exist and undergo spontaneous switching to generate the other subpopulation, remains unclear. Here, we develop a mechanism-based mathematical model that captures the set of experimentally reported interactions among AMPK and Akt. Our simulations suggest that the AMPK-Akt feedback loop can give rise to two co-existing phenotypes (pAkt
/ pAMPK
and pAMPK
/pAkt
) in specific parameter regimes. Next, to test the model predictions, we segregated these two subpopulations in MDA-MB-231 cells and observed that each of them was capable of switching to another in adherent conditions. Finally, the predicted trends are supported by clinical data analysis of The Cancer Genome Atlas (TCGA) breast cancer and pan-cancer cohorts that revealed negatively correlated pAMPK and pAkt protein levels. Overall, our integrated computational-experimental approach unravels that AMPK-Akt feedback loop can generate multi-stability and drive phenotypic switching and heterogeneity in a cancer cell population.
Transformation by the Src tyrosine kinase (Src) promotes nonanchored cell growth and migration. However, nontransformed cells can force Src-transformed cells to assume a normal morphology and ...phenotype by a process called 'contact normalization'. It has become clear that microRNA (miRNA) can affect tumorigenesis by targeting gene products that direct cell growth and migration. However, the roles of miRNA in Src transformation or contact normalization have not yet been reported. We examined the expression of 95 miRNAs and found 9 of them significantly affected by Src. In this study, we report that miR-218 and miR-224 were most significantly induced by Src, but not affected by contact normalization. In contrast, miR-126 was most significantly suppressed by Src and was induced by contact normalization in transformed cells. Mir-126 targets Crk, a component of the focal adhesion network that participates in events required for tumor cell migration. Accordingly, we show that miR-126 expression correlates inversely with Crk levels, motility and the invasive potential of human mammary carcinoma cells. Moreover, we show that miR-224 expression promotes nonanchored growth of nontransformed cells. These data reveal novel insights into how Src regulates miRNA expression to promote hallmarks of tumor cell growth and invasion, and how nontransformed cells can affect miRNA expression in adjacent tumor cells to inhibit this process.
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