MEK/ERK-mediated oncogenic signals promote secretion of extracellular vesicles by controlling lysosome function.

Cancer cells secrete large amounts of extracellular vesicles (EVs) originating from multivesicular bodies (MVBs). Mature MVBs fuse either with the plasma membrane for release as EVs, often referred as to exosomes or with lysosomes for degradation. However, the mechanisms regulating MVB fate remain unknown. Here, we investigated the regulators of MVB fate by analyzing the effects of signaling inhibitors on EV secretion from cancer cells engineered to secrete luciferase-labeled EVs. Inhibition of the oncogenic MEK/ERK pathway suppressed EV release and activated lysosome formation. MEK/ERK-mediated lysosomal inactivation impaired MVB degradation, resulting in increased EV secretion from cancer cells. Moreover, MEK/ERK inhibition prevented c-MYC expression and induced the nuclear translocation of MiT/TFE transcription factors, thereby promoting the activation of lysosome-related genes, including the gene encoding a subunit of vacuolar-type H+ -ATPase, which is responsible for lysosomal acidification and function. Furthermore, c-MYC upregulation was associated with lysosomal gene downregulation in MEK/ERK-activated renal cancer cells/tissues. These findings suggest that the MEK/ERK/c-MYC pathway controls MVB fate and promotes EV production in human cancers by inactivating lysosomal function.

SNAP23-Mediated Perturbation of Cholesterol-Enriched Membrane Microdomain Promotes Extracellular Vesicle Production in Src-Activated Cancer Cells.

Extracellular vesicles (EVs) originating from intraluminal vesicles (ILVs) formed within multivesicular bodies (MVBs), often referred to as small EV (sEV) or exosomes, are aberrantly produced by cancer cells and regulate the tumor microenvironment. The tyrosine kinase c-Src is upregulated in a wide variety of human cancers and is involved in promoting sEV secretion, suggesting its role in malignant progression. In this study, we found that activated Src liberated synaptosomal-associated protein 23 (SNAP23), a SNARE molecule, from lipid rafts to non-rafts on cellular membrane. We also demonstrated that SNAP23 localized in non-rafts induced cholesterol downregulation and ILV formation, resulting in the upregulation of sEV production in c-Src-transformed cells. Furthermore, the contribution of the SNAP23-cholesterol axis on sEV upregulation was confirmed in pancreatic cancer cells. High SNAP23 expression is associated with poor prognosis in patients with pancreatic cancer. These findings suggest a unique mechanism for the upregulation of sEV production via SNAP23-mediated cholesterol downregulation in Src-activated cancer cells.

c-Src promotes tumor progression through downregulation of microRNA-129-1-3p.

MicroRNAs (miRNAs) fine-tune cellular signaling by regulating expression of signaling proteins, and aberrant expression of miRNAs is observed in many cancers. The tyrosine kinase c-Src is upregulated in various human cancers, but the molecular mechanisms underlying c-Src-mediated tumor progression remain unclear. In previous investigations of miRNA-mediated control of c-Src-related oncogenic pathways, we identified miRNAs that were downregulated in association with c-Src transformation and uncovered the signaling networks by predicting their target genes, which might act cooperatively to control tumor progression. Here, to further elucidate the process of cell transformation driven by c-Src, we analyzed the expression profiles of miRNAs in a doxycycline-inducible Src expression system. We found that miRNA (miR)-129-1-3p was downregulated in the early phase of c-Src-induced cell transformation, and that reexpression of miR-129-1-3p disrupted c-Src-induced cell transformation. In addition, miR-129-1-3p downregulation was tightly associated with tumor progression in human colon cancer cells/tissues. Expression of miR-129-1-3p in human colon cancer cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasion. We also identified c-Src and its critical substrate Fer, and c-Yes, a member of the Src family of kinases, as novel targets of miR-129-1-3p. Furthermore, we found that miR-129-1-3p-mediated regulation of c-Src/Fer and c-Yes is important for controlling cell adhesion and invasion. Downregulation of miR-129-1-3p by early activation of c-Src increases expression of these target genes and synergistically promotes c-Src-related oncogenic signaling. Thus, c-Src-miR-129-1-3p circuits serve as critical triggers for tumor progression in many human cancers that harbor upregulation of c-Src.

Rictor promotes tumor progression of rapamycin-insensitive triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC), characterized by decreased expression of hormone receptors and human epidermal growth factor type 2 receptor, has poor prognosis and lacks effective therapeutics. Recently, the mTOR inhibitor rapamycin and its analogs have attracted growing interests and evaluated as therapeutic agents against TNBC, in which the PI3K/AKT/mTOR pathway is often activated. However, some TNBCs are less sensitive to these drugs. In this study, we found that the sensitivity of TNBC cells to rapamycin was highly dependent on the expression level of rapamycin-insensitive companion of mTOR (Rictor), a key component of the mTOR complex 2. Repression of the Rictor expression strongly suppressed the growth of rapamycin-insensitive tumor cells. Furthermore, we showed that the suppression of Rictor expression was also effective in rapamycin-insensitive cells that had acquired resistance to mTOR kinase inhibitors. These findings indicate that Rictor can be a predictive marker for the use of rapamycin analogs in TNBC and highlight the need to develop therapeutics targeting Rictor in the treatment of TNBC.

MicroRNA-137-mediated Src oncogenic signaling promotes cancer progression.

The tyrosine kinase c-Src is frequently overexpressed and activated in a wide variety of human cancers. However, the molecular mechanisms responsible for the upregulation of c-Src remain elusive. To examine whether microRNA-mediated c-Src upregulation promotes cancer progression, we screened miRNAs with complementarity to the 3'-UTR of c-Src mRNA. Among these miRNAs, down-regulation of miR-137 was tightly associated with c-Src-mediated tumor progression of human colon cancer cells/tissues. Re-expression of miR-137 in human colon cancer cells suppressed tumor growth and caused the disruption of focal contacts, suppression of cell adhesion, and invasion, although restoration of c-Src in miR-137-treated cells could not fully rescue the tumor-suppressive effect of miR-137. We found that miR-137 targets AKT2 and paxillin also and miR-137-mediated regulation of c-Src /AKT2 is crucial for controlling tumor growth, whereas that of c-Src/paxillin contributes to malignancy. miR-137 suppressed Src-related oncogenic signaling and changed the expression of miRNAs that are regulated by Src activation. miR-137 controls the expression of c-Src/AKT2/paxillin and synergistically suppresses Src oncogenic signaling evoked from focal adhesions. In various human cancers that harbor c-Src upregulation, the dysfunction of this novel mechanism would serve as a critical trigger for tumor progression.

MicroRNA-27b suppresses tumor progression by regulating ARFGEF1 and focal adhesion signaling.

The non-receptor tyrosine kinase c-Src is frequently activated during progression of colon cancers. In this study, we found that among the c-Src-regulated microRNAs (miRNAs), miR-27b is also repressed by activation of K-Ras/H-Ras. Inhibitor studies suggested that the phosphatidylinositol 3-kinase pathway is involved in the repression of miR-27b. MicroRNA-27b was repressed in various colon cancer cell lines and tumor tissues. Re-expression of miR-27b in human colon cancer HCT116 cells caused morphological changes and suppressed tumor growth, cell adhesion, and invasion. We also identified ARFGEF1 and paxillin as novel targets of miR-27b, and found that miR-27b-mediated regulation of ARFGEF1 is crucial for controlling anchorage-independent growth, and that of paxillin is important for controlling cell adhesion and invasion. Re-expression of miR-27b suppressed the activation of c-Src induced by integrin-mediated cell adhesion, suggesting that repression of miR-27b may contribute to c-Src activation in cancer cells. These findings show that miR-27b functions as a tumor suppressor by controlling ARFGEF1 and the paxillin/c-Src circuit at focal adhesions.

The lipid raft-anchored adaptor protein Cbp controls the oncogenic potential of c-Src.

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis. Cbp-deficient cells are more susceptible to v-Src transformation than their parental cells. Upon phosphorylation, Cbp specifically binds to activated c-Src and sequesters it in lipid rafts, resulting in an efficient suppression of c-Src function independent of Csk. In some human cancer cells and tumors, Cbp is downregulated and the introduction of Cbp significantly suppresses tumorigenesis. These findings indicate a potential role for Cbp as a suppressor of c-Src-mediated tumor progression.

The lysosomal signaling anchor p18/LAMTOR1 controls epidermal development by regulating lysosome-mediated catabolic processes.

The lysosomal adaptor protein p18 is an essential anchor of a scaffolding complex for the mTORC1 and MAPK pathways, which play crucial roles in controlling cell growth and energy homeostasis. To elucidate the in vivo function of the p18-mediated pathway, we conditionally ablated p18 in the mouse epidermis. Mutant mice were born with severe defects in formation of the stratum corneum and died within 12 h after birth due to dehydration caused by loss of skin barrier function. Mutant epidermal cells can grow and differentiate into granular cells, but exhibit functional defects in corneocyte maturation. Electron microscopy identified abnormal immature cells, overlying the mutant granular cells, which accumulated autophagosomes, glycogen granules and dead nuclei. Cell culture analysis showed that loss of p18 attenuated lysosome function, resulting in accumulation of immature lysosomes and autophagosomes. Analyses of lysosome behavior revealed that p18 is required for functional interaction between lysosomes and target organelles including autophagosomes. These findings suggest that p18-mediated pathways control lysosome-mediated catabolic processes, which are crucial for the development of mouse epidermis.

c-Src-induced activation of ceramide metabolism impairs membrane microdomains and promotes malignant progression by facilitating the translocation of c-Src to focal adhesions.

The proto-oncogenic tyrosine kinase c-Src is up-regulated in various human cancers, implicating its role in tumour progression. Upon activation, c-Src translocates to focal adhesions and initiates intracellular signalling cascades that promote malignant transformation, but the underlying mechanisms for c-Src translocation remain unclear. In the present study we show that c-Src up-regulation perturbs sphingolipid/cholesterol-enriched membrane microdomains by activating ceramide synthesis, resulting in promotion of c-Src translocation. Using an inducible c-Src expression system in Csk (C-terminal Src kinase)-deficient fibroblasts, we found that translocation of c-Src to focal adhesions/podosomes occurs in the later stages of cell transformation. Activated c-Src is liberated from microdomains and promotes the phosphorylation of FAK (focal adhesion kinase) and cortactin localized to focal adhesions/podosomes. In parallel with these events, anabolic metabolism of ceramides is activated by up-regulation of the de novo synthesis pathway. Inhibition of ceramide conversion into glucosylceramide promotes liberation of c-Src from microdomains, and inhibition of de novo ceramide synthesis restores the microdomain distribution of c-Src and suppresses malignant phenotypes such as increased cell motility and anchorage-independent cell growth. These results suggest that c-Src-induced activation of ceramide synthesis impairs the integrity of microdomains and contributes to malignant progression by promoting the translocation of c-Src to focal adhesions/podosomes.

Identification of a new interaction mode between the Src homology 2 domain of C-terminal Src kinase (Csk) and Csk-binding protein/phosphoprotein associated with glycosphingolipid microdomains.

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the ßB/ßC loop of the SH2 domain.

Dissection of Wnt5a-Ror2 signaling leading to matrix metalloproteinase (MMP-13) expression.

It has been shown that constitutively active Wnt5a-Ror2 signaling in osteosarcoma cell lines plays crucial roles in induced expression of matrix metalloproteinase-13 (MMP-13), required for their invasiveness; however, it remains largely unclear about the molecular basis of MMP-13 gene induction by Wnt5a-Ror2 signaling. Here we show by reporter assay that the activator protein 1 (AP1) (binding site in the promoter region of MMP-13 gene is primarily responsible for its transcriptional activation by Wnt5a-Ror2 signaling in osteosarcoma cell lines SaOS-2 and U2OS. Chromatin immunoprecipitation assays revealed that c-Jun and ATF2 are crucial transcription factors recruited to the AP1-binding site in the MMP-13 gene promoter during Wnt5a-Ror2 signaling in SaOS-2 cells. Using siRNA-mediated suppression or specific inhibitors, we also show that Dishevelled2 (Dvl2) and c-Jun N-terminal kinase are required for MMP-13 gene induction presumably via phosphorylation of c-Jun and ATF2 during Wnt5a-Ror2 signaling in SaOS-2 cells. Interestingly, Dvl2 and Rac1, but not Dvl3, are required for MMP-13 expression in SaOS-2 cells, whereas Dvl3, but not Dvl2 and Rac1, is required for its expression in U2OS cells, indicating the presence of distinct intracellular signaling machineries leading to expression of the same gene, in this case MMP-13 gene in different osteosarcoma cell lines. Moreover, we provide evidence suggesting that Wnt5a-Ror2 signaling might also be required for expression of MMP-13 gene during the development of the cartilaginous tissue.

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones.

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti-ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C-terminal src kinase)-binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent-resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti-ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over-expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314-phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314-phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP-43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.

The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation.

Podosomes and invadopodia are actin-rich membrane protrusions that play a crucial role in cell adhesion and migration, and extracellular matrix remodeling in normal and cancer cells. The formation of podosomes and invadopodia is promoted by upregulation of some oncogenic molecules and is closely related to the invasive potential of cancer cells. However, the molecular mechanisms underlying the podosome and invadopodium formation still remain unclear. Here, we show that a guanine nucleotide exchange factor (GEF) for Rho family GTPases (Arhgef5) is crucial for Src-induced podosome formation. Using an inducible system for Src activation, we found that Src-induced podosome formation depends upon the Src SH3 domain, and identified Arhgef5 as a Src SH3-binding protein. RNA interference (RNAi)-mediated depletion of Arhgef5 caused robust inhibition of Src-dependent podosome formation. Overexpression of Arhgef5 promoted actin stress fiber remodeling through activating RhoA, and the activation of RhoA or Cdc42 was required for Src-induced podosome formation. Arhgef5 was tyrosine-phosphorylated by Src and bound to Src to positively regulate its activity. Furthermore, the pleckstrin homology (PH) domain of Arhgef5 was required for podosome formation, and Arhgef5 formed a ternary complex with Src and phosphoinositide 3-kinase when Src and/or Arhgef5 were upregulated. These findings provide novel insights into the molecular mechanisms of podosome and invadopodium formation induced by Src upregulation.

Down-regulation of the tumor suppressor C-terminal Src kinase (Csk)-binding protein (Cbp)/PAG1 is mediated by epigenetic histone modifications via the mitogen-activated protein kinase (MAPK)/phosphatidylinositol 3-kinase (PI3K) pathway.

The transmembrane adaptor protein Cbp (or PAG1) functions as a suppressor of Src-mediated tumor progression by promoting the inactivation of Src. The expression of Cbp is down-regulated in Src-transformed cells and in various human cancer cells, suggesting a potential role for Cbp as a tumor suppressor. However, the mechanisms underlying the down-regulation of Cbp remain unknown. The present study shows that Cbp expression is down-regulated by epigenetic histone modifications via the MAPK/PI3K pathway. In mouse embryonic fibroblasts, transformation by oncogenic Src and Ras induced a marked down-regulation of Cbp expression. The levels of Cbp expression were inversely correlated with the activity of MEK and Akt, and Cbp down-regulation was suppressed by inhibiting MEK and PI3K. Src transformation did not affect the stability of Cbp mRNA, the transcriptional activity of the cbp promoter, or the DNA methylation status of the cbp promoter CpG islands. However, Cbp expression was restored by treatment with histone deacetylase (HDAC) inhibitors and by siRNA-mediated knockdown of HDAC1/2. Src transformation significantly decreased the acetylation levels of histone H4 and increased the trimethylation levels of histone H3 lysine 27 in the cbp promoter. EGF-induced Cbp down-regulation was also suppressed by inhibiting MEK and HDAC. Furthermore, the inhibition of MEK or HDAC restored Cbp expression in human cancer cells harboring Cbp down-regulation through promoter hypomethylation. These findings suggest that Cbp down-regulation is primarily mediated by epigenetic histone modifications via oncogenic MAPK/PI3K pathways in a subset of cancer cells.

The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes.

The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

Quantification and Imaging of Exosomes via Luciferase-Fused Exosome Marker Proteins: ExoLuc System.

Bioluminescence (BL) has been widely used to quantitatively monitor various biological phenomena. Here, we describe a protocol for preparing and using cells expressing exosomes labeled with luciferase. The BL of the culture medium of these cells is proportional to the number of secreted exosome particles obtained by well-established nanoparticle tracking analysis, allowing easy, rapid, and sensitive quantification of exosomes in vitro and in vivo. This method, designated the ExoLuc system, is a powerful tool for analyzing the molecular mechanisms of exosome biosynthesis, secretion, uptake, and biodistribution.

Asteltoxin inhibits extracellular vesicle production through AMPK/mTOR-mediated activation of lysosome function.

Cancer cells secrete aberrantly large amounts of extracellular vesicles (EVs) including exosomes, which originate from multivesicular bodies (MVBs). Because EVs potentially contribute to tumor progression, EV inhibitors are of interest as novel therapeutics. We screened a fungal natural product library. Using cancer cells engineered to secrete luciferase-labeled EVs, we identified asteltoxin, which inhibits mitochondrial ATP synthase, as an EV inhibitor. Low concentrations of asteltoxin inhibited EV secretion without inducing mitochondrial damage. Asteltoxin attenuated cellular ATP levels and induced AMPK-mediated mTORC1 inactivation. Consequently, MiT/TFE transcription factors are translocated into the nucleus, promoting transcription of lysosomal genes and lysosome activation. Electron microscopy analysis revealed that the number of lysosomes increased relative to that of MVBs and the level of EVs decreased after treatment with asteltoxin or rapamycin, an mTORC1 inhibitor. These findings suggest that asteltoxin represents a new type of EV inhibitor that controls MVB fate.

A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation.

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry). Using this method, we reveal a severe skewing in acyl chains in phosphoinositides in Pten-deficient prostate cancer tissues, extracellular mobilization of phosphoinositides upon expression of oncogenic PIK3CA, and a unique profile for exosomal phosphoinositides. Thus, our approach allows characterizing the dynamics of phosphoinositide acyl variants in intracellular and extracellular milieus.

Metabolomics of small extracellular vesicles derived from isocitrate dehydrogenase 1-mutant HCT116 cells collected by semi-automated size exclusion chromatography.

Cancer-derived small extracellular vesicles (sEVs) are multifunctional particles with a lipid bilayer structure that are involved in cancer progression, such as malignant proliferation, distant metastasis, and cancer immunity evasion. The separation protocol used to isolate sEVs is an important process and thus, several have been developed, including ultracentrifugation (UC), size exclusion chromatography (SEC), and affinity purification using antibodies against sEV surface antigens. However, the effects of different separation methods on sEV components have not been adequately examined. Here, we developed a semi-automated system for collecting sEVs by combining SEC and preparative high-performance liquid chromatography and applied it to metabolome analysis. The developed SEC system could recover sEVs more efficiently and non-destructively than UC, suggesting that it is an appropriate recovery method for metabolic analysis and reflects biological conditions. Furthermore, using the developed SEC system, we performed metabolome analysis of sEVs from isocitrate dehydrogenase 1 (IDH)-mutated human colon HCT116 cells, which produce the oncogenic metabolite, 2-hydroxyglutaric acid (2-HG). IDH1-mutated HCT116 cells released significantly more sEVs than wild-type (WT) cells. The metabolomic profiles of IDH1 mutant and WT cells showed distinct differences between the cells and their sEVs. Notably, in IDH mutant cells, large amounts of 2-HG were detected not only in cells, but also in sEVs. These results indicate that the SEC system we developed has wide potential applications in sEVs research.

Purvalanol A, a CDK inhibitor, effectively suppresses Src-mediated transformation by inhibiting both CDKs and c-Src.

The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However, Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression, we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src. We found that purvalanol A, developed as a CDK inhibitor, potently suppressed the anchorage-independent growth of c-Src-transformed cells, indicating that the activation of CDKs contributes to the c-Src transformation. We also found that purvalanol A suppressed the c-Src activity as effectively as the Src-selective inhibitor PP2, and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2-M arrest, whereas PP2 weakly acted on the G1-S transition. Furthermore, when compared with PP2, purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells, in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi-selective purvalanol A is effective in suppressing cancer progression associated with c-Src up-regulation.