Implication of integrin α2ß1 in senescence of SK-Mel-147 human melanoma cells.

This investigation addressed the impact of integrin-initiated signaling pathways on senescence of tumor cells. In a model of human SK-Mel-147 melanoma cells, the silencing of integrin α2ß1 strongly reduced cell proliferation and enhanced the percentage of SA-ß-Gal-positive cells, a phenotypic feature of cellular senescence. These changes were accompanied by a significant increase in the activity of Akt and mTOR protein kinases and also in the expression of p53 and p21 oncosuppressors. Pharmacological inhibition of Akt and mTORC1 and genetic inhibition of p53 and p21 reduced the senescence of α2ß1-depleted SK-Mel-147 cells to the level of control cells. Based on our earlier data on the non-canonical functions of Akt isomers in the invasion and anoikis of SK-Mel-147 cells, we investigated the role of Akt isomers in senescence induced by α2ß1 suppression. The inhibition of Akt1 strongly reduced the percentage of SA-ß-Gal-positive cells in the α2ß1-depleted cell population, while the inhibition of Akt2 did not have a noticeable effect. Our data demonstrated for the first time that α2ß1 is involved in the protection of tumor cells against senescence and that senescence, which is induced by the downregulation of α2ß, is based on a signaling mechanism in which Akt1 performs a non-canonical function.

Implication of integrins α3ß1 and α5ß1 in invasion and anoikis of SK-Mel-147 human melanoma cells: non-canonical functions of protein kinase Akt.

Downregulation of integrins α3ß1 and α5ß1 strongly decreased cell colony formation and in vitro invasion and markedly enhanced anoikis in SK-Mel-147 human melanoma cells. These modifications were accompanied by a marked increase in the levels of active Akt protein kinase, which indicated it played a non-canonical function in the melanoma cells. Pharmacological inhibition of Akt1, an Akt isozyme, in cells depleted of α3ß1 or α5ß1 restored their invasive activity, while inhibition of the Akt 2 isoform did not cause a visible effect. Similar to our previous results with the α2ß1 integrin, this finding suggested that in signaling pathways initiated by α3ß1 and α5ß1, the Akt1 isoform performs a non-canonical function in regulating invasive phenotype of melanoma cells. In contrast, when the effects of Akt inhibitors on anoikis of the melanoma cells were compared, the Akt2 isoform demonstrated a non-canonical activity in which Akt2 suppression led to a significant attenuation of apoptosis in cells with downregulated α3ß1 or α5ß1. Our results were the first evidence that, in the same tumor cells, different integrins can control various manifestations of tumor progression through distinct signaling pathways that are both common to various integrins and specific to a particular receptor.

Implication of integrin α2ß1 in anoikis of SK-Mel-147 human melanoma cells: a non-canonical function of Akt protein kinase.

Suppression of anoikis, a kind of apoptosis caused by disruption of contacts between cell and extracellular matrix, is an important prerequisite for cancer cell metastasis. In this communication, we demonstrate that shRNA-mediated depletion of α2 integrin subunit induces anoikis and substantially decreases colony-forming potential in SK-Mel-147 human melanoma cells. Suppression of α2ß1 upregulates the levels of pro-apoptotic protein p53 and cyclin-dependent kinase inhibitors p21 and p27. Concomitantly, we detected decrease in the levels of anti-apoptotic protein Bcl-2 and cell cycle regulator c-Myc. Moreover, depletion of α2ß1 reduces the activity of protein kinase Erk, while increases activity of Akt kinase. Pharmacological inhibition of P3IK kinase, an upstream activator of Akt, greatly enhanced anoikis in control cells while reduced that in cells with decreased levels of α2ß1. Of three isoforms of Akt, down-regulation of Akt1 greatly diminished anoikis of cells depleted of α2ß1, while down-regulation of Akt2 and Akt3 sharply increased anoikis in these cells. These findings were supported by the data of pharmacological inhibition of the Akt isoforms. Our results demonstrate for the first time that anoikis induced by α2ß1 integrin knockdown can be attenuated by Akt1 inhibition.

KLF9-dependent ROS regulate melanoma progression in stage-specific manner.

Although antioxidants promote melanoma metastasis, the role of reactive oxygen species (ROS) in other stages of melanoma progression is controversial. Moreover, genes regulating ROS have not been functionally characterized throughout the entire tumor progression in mouse models of cancer. To address this question, we crossed mice-bearing knock-out of Klf9, an ubiquitous transcriptional regulator of oxidative stress, with two conditional melanocytic mouse models: BrafCA mice, where BrafV600E causes premalignant melanocytic hyperplasia, and BrafCA/Pten-/- mice, where BrafV600E and loss of Pten induce primary melanomas and metastases. Klf9 deficiency inhibited premalignant melanocytic hyperplasia in BrafCA mice but did not affect formation and growth of BrafCA/Pten-/- primary melanomas. It also, as expected, promoted BrafCA/Pten-/- metastasis. Treatment with antioxidant N-acetyl cysteine phenocopied loss of Klf9 including suppression of melanocytic hyperplasia. We were interested in a different role of Klf9 in regulation of cell proliferation in BrafCA and BrafCA/Pten-/- melanocytic cells. Mechanistically, we demonstrated that BRAFV600E signaling transcriptionally upregulated KLF9 and that KLF9-dependent ROS were required for full-scale activation of ERK1/2 and induction of cell proliferation by BRAFV600E. PTEN depletion in BRAFV600E-melanocytes did not further activate ERK1/2 and cell proliferation, but rendered these phenotypes insensitive to KLF9 and ROS. Our data identified an essential role of KLF9-dependent ROS in BRAFV600E signaling in premalignant melanocytes, offered an explanation to variable role of ROS in premalignant and transformed melanocytic cells and suggested a novel mechanism for suppression of premalignant growth by topical antioxidants.

FOXQ1 controls the induced differentiation of melanocytic cells.

Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAFV600E-dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.

Melanoma Suppressor Functions of the Carcinoma Oncogene FOXQ1.

Lineage-specific regulation of tumor progression by the same transcription factor is understudied. We find that levels of the FOXQ1 transcription factor, an oncogene in carcinomas, are decreased during melanoma progression. Moreover, in contrast to carcinomas, FOXQ1 suppresses epithelial-to-mesenchymal transition, invasion, and metastasis in melanoma cells. We find that these lineage-specific functions of FOXQ1 largely depend on its ability to activate (in carcinomas) or repress (in melanoma) transcription of the N-cadherin gene (CDH2). We demonstrate that FOXQ1 interacts with nuclear ß-catenin and TLE proteins, and the ß-catenin/TLE ratio, which is higher in carcinoma than melanoma cells, determines the effect of FOXQ1 on CDH2 transcription. Accordingly, other FOXQ1-dependent phenotypes can be manipulated by altering nuclear ß-catenin or TLE proteins levels. Our data identify FOXQ1 as a melanoma suppressor and establish a mechanism underlying its inverse lineage-specific transcriptional regulation of transformed phenotypes.

A purine nucleotide biosynthesis enzyme guanosine monophosphate reductase is a suppressor of melanoma invasion.

Melanoma is one of the most aggressive types of human cancers, and the mechanisms underlying melanoma invasive phenotype are not completely understood. Here, we report that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in de novo biosynthesis of purine nucleotides, was downregulated in the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR downregulates the amounts of several GTP-bound (active) Rho-GTPases and suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade in vitro, and grow as tumor xenografts in vivo. Mechanistically, we demonstrated that GMPR partially depletes intracellular GTP pools. Pharmacological inhibition of de novo GTP biosynthesis suppressed whereas addition of exogenous guanosine increased invasion of melanoma cells as well as cells from other cancer types. Our data identify GMPR as a melanoma invasion suppressor and establish a link between guanosine metabolism and Rho-GTPase-dependent melanoma cell invasion.

Ribonucleotide reductase and thymidylate synthase or exogenous deoxyribonucleosides reduce DNA damage and senescence caused by C-MYC depletion.

The down-regulation of dominant oncogenes, including C-MYC, in tumor cells often leads to the induction of senescence via mechanisms that are not completely identified. In the current study, we demonstrate that MYC-depleted melanoma cells undergo extensive DNA damage that is caused by the underexpression of thymidylate synthase (TS) and ribonucleotide reductase (RR) and subsequent depletion of deoxyribonucleoside triphosphate pools. Simultaneous genetic inhibition of TS and RR in melanoma cells induced DNA damage and senescence phenotypes very similar to the ones caused by MYC-depletion. Reciprocally, overexpression of TS and RR in melanoma cells or addition of deoxyribo-nucleosides to culture media substantially inhibited DNA damage and senescence-associated phenotypes caused by C-MYC depletion. Our data demonstrate the essential role of TS and RR in C-MYC-dependent suppression of senescence in melanoma cells.

Integrin α5ß1 simultaneously controls EGFR-dependent proliferation and Akt-dependent pro-survival signaling in epidermoid carcinoma cells.

To delineate distinctive role of the components of α5ß1 integrin-EGFR axis in control of epidermoid carcinoma cell proliferation, we performed individual inhibition of α5ß1 and EGFR via genetic and phamacological methods, respectively. We demonstrated that pharmacological inhibition of epidermal growth factor receptor (EGFR) significantly affected proliferation of A431 human cells by inducing the G0/G1 cell cycle arrest, whereas shRNA-mediated depletion of α5 subunit of α5ß1 integrin led to a similar type of cell cycle arrest followed by significant apoptosis. Both treatments resulted in suppression of activated (phosphorylated) forms of focal adhesion kinase (FAK) and Erk. However, unlike EGFR inhibition, depletion of α5 led to substantial suppression of AKT activity. Accordingly, pharmacological inhibition of EGFR and AKT recapitulated detrimental effects caused by shRNA-mediated depletion of α5. Moreover, depletion of α5 led to a severe drop in the amounts of active EGFR. Thus, for the first time, we demonstrated that α5ß1 integrin simultaneously maintains pro-survival signaling via continuous activation of AKT and up-regulates proliferation via activation of EGFR.

Recent progress in genetics of aging, senescence and longevity: focusing on cancer-related genes.

It is widely believed that aging results from the accumulation of molecular damage, including damage of DNA and mitochondria and accumulation of molecular garbage both inside and outside of the cell. Recently, this paradigm is being replaced by the "hyperfunction theory", which postulates that aging is caused by activation of signal transduction pathways such as TOR (Target of Rapamycin). These pathways consist of different enzymes, mostly kinases, but also phosphatases, deacetylases, GTPases, and some other molecules that cause overactivation of normal cellular functions. Overactivation of these sensory signal transduction pathways can cause cellular senescence, age-related diseases, including cancer, and shorten life span. Here we review some of the numerous very recent publications on the role of signal transduction molecules in aging and age-related diseases. As was emphasized by the author of the "hyperfunction model", many (or actually all) of them also play roles in cancer. So these "participants" in pro-aging signaling pathways are actually very well acquainted to cancer researchers. A cancer-related journal such as Oncotarget is the perfect place for publication of such experimental studies, reviews and perspectives, as it can bridge the gap between cancer and aging researchers.

Fucans, but not fucomannoglucuronans, determine the biological activities of sulfated polysaccharides from Laminaria saccharina brown seaweed.

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed.

A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds.

The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans obtained from nine species of brown algae were studied in order to examine the influence of fucoidan origin and composition on their biological activities. All fucoidans inhibited leucocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate nor other structural features of their polysaccharide backbones significantly affected the efficacy of fucoidans in this model. In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that only polysaccharides from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, and Ascophyllum nodosum could serve as P-selectin inhibitors. All fucoidans, except that from Cladosiphon okamuranus carrying substantial levels of 2-O-alpha-D-glucuronopyranosyl branches in the linear (1-->3)-linked poly-alpha-fucopyranoside chain, exhibited anticoagulant activity as measured by activated partial thromboplastin time whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens displayed strong antithrombin activity in a platelet aggregation test. The last fucoidans potently inhibited human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and this property correlated with decreased levels of plasminogen-activator inhibitor-1 in HUVEC supernatants, suggesting a possible mechanism of fucoidan-induced inhibition of tubulogenesis. Finally, fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumor metastasis. The data presented herein provide a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.

Multidrug-resistant tumor cells with decreased malignancy: a role for integrin alphavbeta3.

We studied whether acquisition of multidrug resistance (MDR) by tumor cells can alter their integrin profile and malignant behavior. Hamster fibroblast cell line HET-SR-2SC-LNM was selected for MDR, yielding the 2SC/20 subline. Compared with the parental cells, the 2SC/20 subline weakly adhered to denatured collagen (dCol) which correlated with decreased expression of alphavbeta3, a dCol receptor. Importantly, 2SC/20 subline demonstrated significantly decreased activity of collagenase MMP-2, lower ability to invade Matrigel, and attenuated metastasis in syngeneic animals. We provide evidence for the first time that selection for MDR can be associated with down-regulation of alphavbeta3 integrin, supporting our recent proof of the pro-apoptotic role of this integrin (Oncogene 20 (2001) 4710). Lack of alphavbeta3 expression may link cell survival under toxic conditions with decreased malignancy of the resulting drug resistant tumor.