FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells.

To exploit vulnerabilities of tumors, it is urgent to identify associated defects in genome maintenance. One unsolved problem is the mechanism of regulation of DNA double-strand break repair by REV7 in complex with 53BP1 and RIF1, and its influence on repair pathway choice between homologous recombination and non-homologous end-joining. We searched for REV7-associated factors in human cells and found FAM35A, a previously unstudied protein with an unstructured N-terminal region and a C-terminal region harboring three OB-fold domains similar to single-stranded DNA-binding protein RPA, as novel interactor of REV7/RIF1/53BP1. FAM35A re-localized in damaged cell nuclei, and its knockdown caused sensitivity to DNA-damaging agents. In a BRCA1-mutant cell line, however, depletion of FAM35A increased resistance to camptothecin, suggesting that FAM35A participates in processing of DNA ends to allow more efficient DNA repair. We found FAM35A absent in one widely used BRCA1-mutant cancer cell line (HCC1937) with anomalous resistance to PARP inhibitors. A survey of FAM35A alterations revealed that the gene is altered at the highest frequency in prostate cancers (up to 13%) and significantly less expressed in metastatic cases, revealing promise for FAM35A as a therapeutically relevant cancer marker.

USP22 controls multiple signaling pathways that are essential for vasculature formation in the mouse placenta.

USP22, a component of the SAGA complex, is overexpressed in highly aggressive cancers, but the normal functions of this deubiquitinase are not well defined. We determined that loss of USP22 in mice results in embryonic lethality due to defects in extra-embryonic placental tissues and failure to establish proper vascular interactions with the maternal circulatory system. These phenotypes arise from abnormal gene expression patterns that reflect defective kinase signaling, including TGFß and several receptor tyrosine kinase pathways. USP22 deletion in endothelial cells and pericytes that are induced from embryonic stem cells also hinders these signaling cascades, with detrimental effects on cell survival and differentiation as well as on the ability to form vessels. Our findings provide new insights into the functions of USP22 during development that may offer clues to its role in disease states.

Systematic evaluation of RNA-Seq preparation protocol performance.

BACKGROUND: RNA-Seq is currently the most widely used tool to analyze whole-transcriptome profiles. There are numerous commercial kits available to facilitate preparing RNA-Seq libraries; however, it is still not clear how some of these kits perform in terms of: 1) ribosomal RNA removal; 2) read coverage or recovery of exonic vs. intronic sequences; 3) identification of differentially expressed genes (DEGs); and 4) detection of long non-coding RNA (lncRNA). In RNA-Seq analysis, understanding the strengths and limitations of commonly used RNA-Seq library preparation protocols is important, as this technology remains costly and time-consuming. RESULTS: In this study, we present a comprehensive evaluation of four RNA-Seq kits. We used three standard input protocols: Illumina TruSeq Stranded Total RNA and mRNA kits, a modified NuGEN Ovation v2 kit, and the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5' and 3' end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes. CONCLUSIONS: At the manufacturers' recommended input RNA levels, all the RNA-Seq library preparation protocols evaluated were suitable for distinguishing between experimental groups, and the TruSeq Stranded mRNA kit was universally applicable to studies focusing on protein-coding gene profiles. The TruSeq protocols tended to capture genes with higher expression and GC content, whereas the modified NuGEN protocol tended to capture longer genes. The SMARTer Ultra Low RNA Kit may be a good choice at the low RNA input level, although it was inferior to the TruSeq mRNA kit at standard input level in terms of rRNA removal, exonic mapping rates and recovered DEGs. Therefore, the choice of RNA-Seq library preparation kit can profoundly affect data outcomes. Consequently, it is a pivotal parameter to consider when designing an RNA-Seq experiment.

Concise Review: NANOG in Cancer Stem Cells and Tumor Development: An Update and Outstanding Questions.

The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers. Functional studies have provided strong evidence that NANOG possesses protumorigenic attributes. In addition to promoting self-renewal and long-term proliferative potential of stem-like cancer cells, NANOG-mediated oncogenic reprogramming may underlie clinical manifestations of malignant disease. In this review, we examine the molecular origin, expression, biological activities, and mechanisms of action of NANOG in various malignancies. We also consider clinical implications such as correlations between NANOG expression and cancer prognosis and/or response to therapy. We surmise that NANOG potentiates the molecular circuitry of tumorigenesis, and thus may represent a novel therapeutic target or biomarker for the diagnosis, prognosis, and treatment outcome of cancer. Finally, we present critical pending questions relating NANOG to cancer stem cells and tumor development.

A direct blood polymerase chain reaction approach for the determination of GP.Mur (Mi.III) and other Hil+ Miltenberger glycophorin variants.

BACKGROUND: GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. STUDY DESIGN AND METHODS: To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. RESULTS: Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing. CONCLUSION: This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.

Andrographolide exhibits anti-invasive activity against colon cancer cells via inhibition of MMP2 activity.

Andrographolide, a major constituent of Andrographis paniculata, was previously shown to exhibit anti-inflammatory, antiviral, and anticancer activities. The anticancer activity of andrographolide includes growth suppression, apoptosis promotion, antiangiogenesis, and antitransformation. However, the effect of andrographolide on cancer metastasis, the most malignant feature of cancer, has not been elucidated extensively. In the present study, we demonstrated that andrographolide at nontoxic to subtoxic concentrations (0.3-3 µM) suppressed the invasion ability of CT26 cells in Matrigel-based invasion assays. In addition, the expression of cell adhesion regulators (ß-catenin and ILK) was not altered by andrographolide treatment. However, andrographolide indeed inhibited matrix metalloproteinase 2 (MMP2) activity without affecting its expression. Furthermore, the activation of ERK, but not Akt, was attenuated by andrographolide treatment. Notably, a similar inhibitory effect of andrographolide on the invasion and MMP2 activity of the human colon cancer cell line HT29 was also observed. In summary, our results indicate that andrographolide exhibits anti-invasive activity against colon cancer cells via inhibition of MMP2 activity.

Intron retention is a hallmark and spliceosome represents a therapeutic vulnerability in aggressive prostate cancer.

The role of dysregulation of mRNA alternative splicing (AS) in the development and progression of solid tumors remains to be defined. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) evolution. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent genomic copy number variations (CNVs), leading to mis-expression of ~68% of SRGs during PCa development and progression. Consequently, many SRGs are prognostic. Surprisingly, androgen receptor controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and inhibits castration-resistant PCa (CRPC) in xenograft and autochthonous PCa models. Altogether, our studies establish aberrant AS landscape caused by dysregulated SRGs as a hallmark of PCa aggressiveness and the spliceosome as a therapeutic vulnerability for CRPC.

Author Correction: LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ATG5 cancer mutations and alternative mRNA splicing reveal a conjugation switch that regulates ATG12-ATG5-ATG16L1 complex assembly and autophagy.

Autophagy is critical for maintaining cellular homeostasis during times of stress, and is thought to play important roles in both tumorigenesis and tumor cell survival. Formation of autophagosomes, which mediate delivery of cytoplasmic cargo to lysosomes, requires multiple autophagy-related (ATG) protein complexes, including the ATG12-ATG5-ATG16L1 complex. Herein, we report that a molecular ATG5 "conjugation switch", comprised of competing ATG12 and ubiquitin conjugation reactions, integrates ATG12-ATG5-ATG16L1 complex assembly with protein quality control of its otherwise highly unstable subunits. This conjugation switch is tightly regulated by ATG16L1, which binds to free ATG5 and mutually protects both proteins from ubiquitin conjugation and proteasomal degradation, thereby instead promoting the irreversible conjugation of ATG12 to ATG5. The resulting ATG12-ATG5 conjugate, in turn, displays enhanced affinity for ATG16L1 and thus fully stabilizes the ATG12-ATG5-ATG16L1 complex. Most importantly, we find in multiple tumor types that ATG5 somatic mutations and alternative mRNA splicing specifically disrupt the ATG16L1-binding pocket in ATG5 and impair the essential ATG5-ATG16L1 interactions that are initially required for ATG12-ATG5 conjugation. Finally, we provide evidence that ATG16L2, which is overexpressed in several cancers relative to ATG16L1, hijacks the conjugation switch by competing with ATG16L1 for binding to ATG5. While ATG16L2 stabilizes ATG5 and enables ATG12-ATG5 conjugation, this endogenous dominant-negative inhibitor simultaneously displaces ATG16L1, resulting in its proteasomal degradation and a block in autophagy. Thus, collectively, our findings provide novel insights into ATG12-ATG5-ATG16L1 complex assembly and reveal multiple mechanisms wherein dysregulation of the ATG5 conjugation switch inhibits autophagy.

LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

LRIG1 has been reported to be a tumor suppressor in gastrointestinal tract and epidermis. However, little is known about the expression, regulation and biological functions of LRIG1 in prostate cancer (PCa). We find that LRIG1 is overexpressed in PCa, but its expression correlates with better patient survival. Functional studies reveal strong tumor-suppressive functions of LRIG1 in both AR+ and AR- xenograft models, and transgenic expression of LRIG1 inhibits tumor development in Hi-Myc and TRAMP models. LRIG1 also inhibits castration-resistant PCa and exhibits therapeutic efficacy in pre-established tumors. We further show that 1) AR directly transactivates LRIG1 through binding to several AR-binding sites in LRIG1 locus, and 2) LRIG1 dampens ERBB expression in a cell type-dependent manner and inhibits ERBB2-driven tumor growth. Collectively, our study indicates that LRIG1 represents a pleiotropic AR-regulated feedback tumor suppressor that functions to restrict oncogenic signaling from AR, Myc, ERBBs, and, likely, other oncogenic drivers.

Linking prostate cancer cell AR heterogeneity to distinct castration and enzalutamide responses.

Expression of androgen receptor (AR) in prostate cancer (PCa) is heterogeneous but the functional significance of AR heterogeneity remains unclear. Screening ~200 castration-resistant PCa (CRPC) cores and whole-mount sections (from 89 patients) reveals 3 AR expression patterns: nuclear (nuc-AR), mixed nuclear/cytoplasmic (nuc/cyto-AR), and low/no expression (AR-/lo). Xenograft modeling demonstrates that AR+ CRPC is enzalutamide-sensitive but AR-/lo CRPC is resistant. Genome editing-derived AR+ and AR-knockout LNCaP cell clones exhibit distinct biological and tumorigenic properties and contrasting responses to enzalutamide. RNA-Seq and biochemical analyses, coupled with experimental combinatorial therapy, identify BCL-2 as a critical therapeutic target and provide proof-of-concept therapeutic regimens for both AR+/hi and AR-/lo CRPC. Our study links AR expression heterogeneity to distinct castration/enzalutamide responses and has important implications in understanding the cellular basis of prostate tumor responses to AR-targeting therapies and in facilitating development of novel therapeutics to target AR-/lo PCa cells/clones.

Developing a Novel Two-Dimensional Culture System to Enrich Human Prostate Luminal Progenitors that Can Function as a Cell of Origin for Prostate Cancer.

Elucidating the cell of origin of cancer has great significance in stratifying patients into appropriate treatment groups and for developing novel targeted therapies. Early studies demonstrate that only stem-like basal cells in the normal human prostate (NHP) can function as the cell of origin for prostate cancer (PCa). Here, we show that the organoids derived from bulk NHP luminal cells can also be tumorigenically transformed. We further show that the WIT medium, which is used to culture human mammary epithelial progenitor cells, when combined with the ROCK inhibitor, can readily propagate a population of progenitor-like cells from the primary NHP luminal cell isolates. Such functionally defined luminal progenitors can be transformed by distinct sets of genetic perturbations (i.e., AR+AKT/ERG or c-MYC+PTEN knockout) to form tumor glands. Genome-wide RNA-Seq analysis of freshly purified unperturbed human benign prostatic basal and luminal cells and culture-expanded lineage-specific stem/progenitor populations reveals that the luminal progenitors possess a distinct gene expression profile that is greatly enriched in advanced, castration-resistant, and metastatic PCa, and it associates with poor patient survival. The ability of the simple two-dimensional culture system reported herein to greatly enrich NHP progenitor-like cells should facilitate biological and biochemical studies as well as high-throughput screening in these cells and in progenitor-like PCa cells. Stem Cells Translational Medicine 2017;6:748-760.

MicroRNA-141 suppresses prostate cancer stem cells and metastasis by targeting a cohort of pro-metastasis genes.

MicroRNAs play important roles in regulating tumour development, progression and metastasis. Here we show that one of the miR-200 family members, miR-141, is under-expressed in several prostate cancer (PCa) stem/progenitor cell populations in both xenograft and primary patient tumours. Enforced expression of miR-141 in CD44+ and bulk PCa cells inhibits cancer stem cell properties including holoclone and sphere formation, as well as invasion, and suppresses tumour regeneration and metastasis. Moreover, miR-141 expression enforces a strong epithelial phenotype with a partial loss of mesenchymal phenotype. Whole-genome RNA sequencing uncovers novel miR-141-regulated molecular targets in PCa cells including the Rho GTPase family members (for example, CDC42, CDC42EP3, RAC1 and ARPC5) and stem cell molecules CD44 and EZH2, all of which are validated as direct and functionally relevant targets of miR-141. Our results suggest that miR-141 employs multiple mechanisms to obstruct tumour growth and metastasis.

Defining a Population of Stem-like Human Prostate Cancer Cells That Can Generate and Propagate Castration-Resistant Prostate Cancer.

PURPOSE: We have shown that the phenotypically undifferentiated (PSA(-/lo)) prostate cancer cell population harbors long-term self-renewing cancer stem cells (CSC) that resist castration, and a subset of the cells within the PSA(-/lo) population bearing the ALDH(hi)CD44(+)α2ß1(+) phenotype (Triple Marker(+)/TM(+)) is capable of robustly initiating xenograft tumors in castrated mice. The goal of the current project is to further characterize the biologic properties of TM(+) prostate cancer cell population, particularly in the context of initiating and propagating castration-resistant prostate cancer (CRPC). EXPERIMENTAL DESIGN: The in vivo CSC activities were measured by limiting-dilution serial tumor transplantation assays in both androgen-dependent and androgen-independent prostate cancer xenograft models. In vitro clonal, clonogenic, and sphere-formation assays were conducted in cells purified from xenograft and patient tumors. qPCR, Western blot, lentiviral-mediated gene knockdown, and human microRNA arrays were performed for mechanistic studies. RESULTS: By focusing on the LAPC9 model, we show that the TM(+) cells are CSCs with both tumor-initiating and tumor-propagating abilities for CRPC. Moreover, primary patient samples have TM(+) cells, which possess CSC activities in "castrated" culture conditions. Mechanistically, we find that (i) the phenotypic markers are causally involved in CRPC development; (ii) the TM(+) cells preferentially express castration resistance and stem cell-associated molecules that regulate their CSC characteristics; and (iii) the TM(+) cells possess distinct microRNA expression profiles and miR-499-5p functions as an oncomir. CONCLUSIONS: Our results define the TM(+) prostate cancer cells as a population of preexistent stem-like cancer cells that can both mediate and propagate CRPC and highlight the TM(+) cell population as a therapeutic target. Clin Cancer Res; 22(17); 4505-16. ©2016 AACR.

NANOG reprograms prostate cancer cells to castration resistance via dynamically repressing and engaging the AR/FOXA1 signaling axis.

The pluripotency transcription factor NANOG has been implicated in tumor development, and NANOG-expressing cancer cells manifest stem cell properties that sustain tumor homeostasis, mediate therapy resistance and fuel tumor progression. However, how NANOG converges on somatic circuitry to trigger oncogenic reprogramming remains obscure. We previously reported that inducible NANOG expression propels the emergence of aggressive castration-resistant prostate cancer phenotypes. Here we first show that endogenous NANOG is required for the growth of castration-resistant prostate cancer xenografts. Genome-wide chromatin immunoprecipitation sequencing coupled with biochemical assays unexpectedly reveals that NANOG co-occupies a distinctive proportion of androgen receptor/Forkhead box A1 genomic loci and physically interacts with androgen receptor and Forkhead box A1. Integrative analysis of chromatin immunoprecipitation sequencing and time-resolved RNA sequencing demonstrates that NANOG dynamically alters androgen receptor/Forkhead box A1 signaling leading to both repression of androgen receptor-regulated pro-differentiation genes and induction of genes associated with cell cycle, stem cells, cell motility and castration resistance. Our studies reveal global molecular mechanisms whereby NANOG reprograms prostate cancer cells to a clinically relevant castration-resistant stem cell-like state driven by distinct NANOG-regulated gene clusters that correlate with patient survival. Thus, reprogramming factors such as NANOG may converge on and alter lineage-specific master transcription factors broadly in somatic cancers, thereby facilitating malignant disease progression and providing a novel route for therapeutic resistance.

Stem cell and neurogenic gene-expression profiles link prostate basal cells to aggressive prostate cancer.

The prostate gland mainly contains basal and luminal cells constructed as a pseudostratified epithelium. Annotation of prostate epithelial transcriptomes provides a foundation for discoveries that can impact disease understanding and treatment. Here we describe a genome-wide transcriptome analysis of human benign prostatic basal and luminal epithelial populations using deep RNA sequencing. Through molecular and biological characterizations, we show that the differential gene-expression profiles account for their distinct functional properties. Strikingly, basal cells preferentially express gene categories associated with stem cells, neurogenesis and ribosomal RNA (rRNA) biogenesis. Consistent with this profile, basal cells functionally exhibit intrinsic stem-like and neurogenic properties with enhanced rRNA transcription activity. Of clinical relevance, the basal cell gene-expression profile is enriched in advanced, anaplastic, castration-resistant and metastatic prostate cancers. Therefore, we link the cell-type-specific gene signatures to aggressive subtypes of prostate cancer and identify gene signatures associated with adverse clinical features.

Longitudinal tracking of subpopulation dynamics and molecular changes during LNCaP cell castration and identification of inhibitors that could target the PSA-/lo castration-resistant cells.

We have recently demonstrated that the undifferentiated PSA-/lo prostate cancer (PCa) cell population harbors self-renewing long-term tumor-propagating cells that are refractory to castration, thus representing a therapeutic target. Our goals here are, by using the same lineage-tracing reporter system, to track the dynamic changes of PSA-/lo and PSA+ cells upon castration in vitro, investigate the molecular changes accompanying persistent castration, and develop large numbers of PSA-/lo PCa cells for drug screening. To these ends, we treated LNCaP cells infected with the PSAP-GFP reporter with three regimens of castration, i.e., CDSS, CDSS plus bicalutamide, and MDV3100 continuously for up to ~21 months. We observed that in the first ~7 months, castration led to time-dependent increases in PSA-/lo cells, loss of AR and PSA expression, increased expression of cancer stem cell markers, and many other molecular changes. Meanwhile, castrated LNCaP cells became resistant to high concentrations of MDV3100, chemotherapeutic drugs, and other agents. However, targeted and medium-throughput library screening identified several kinase (e.g., IGF-1R, AKT, PI3K/mTOR, Syk, GSK3) inhibitors as well as the BCL2 inhibitor that could effectively sensitize the LNCaP-CRPC cells to killing. Of interest, LNCaP cells castrated for >7 months showed evidence of cyclic changes in AR and the mTOR/AKT signaling pathways potentially involving epigenetic mechanisms. These observations indicate that castration elicits numerous molecular changes and leads to enrichment of PSA-/lo PCa cells. The ability to generate large numbers of PSA-/lo PCa cells should allow future high-throughput screening to identify novel therapeutics that specifically target this population.

Systematic dissection of phenotypic, functional, and tumorigenic heterogeneity of human prostate cancer cells.

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA-/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR+PSA+, AR+PSA-, AR-PSA-, and AR-PSA+ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA-/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA+ versus PSA-/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA-/lo PCa cells possess distinct epigenetic profiles. As the PSA-/lo PCa cell population is heterogeneous, in the second part, we employ two PSA- (Du145 and PC3) and two PSA+ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.

Suppression of v-Src transformation by andrographolide via degradation of the v-Src protein and attenuation of the Erk signaling pathway.

Elevated expression and aberrant activation of the src oncogene are strongly associated with cancer initiation and progression, thereby making Src a promising molecular target for anti-cancer therapy. Through drug screening using a temperature-inducible v-Src-transformed epithelial cell line, we found that andrographolide could suppress v-Src-induced transformation and down-regulate v-Src protein expression. In addition, actin cable dissolution and E-cadherin down-regulation, features of transformed phenotype, are perturbed by andrographolide. Moreover, andrographolide promoted v-Src degradation via a ubiquitin-dependent manner. Although andrographolide treatment altered the tyrosine phosphorylation pattern in v-Src-expressing cells, it did not directly affect the kinase activity of v-Src. Both the Erk and phosphatidylinositol 3-kinase signaling pathways were strongly inhibited in andrographolide-treated v-Src cells. However, only MKK inhibitors (PD98059 and U0126) were able to cause a non-transformed morphology similar to that of andrographolide-treated v-Src cells. Moreover, overexpression of constitutively active MKK1 in v-Src cells blocked andrographolide-mediated morphological inhibition. Interestingly, andrographolide treatment could also reduce the protein level of the c-Src truncation mutant (Src531), an Src mutant originally identified from human colon cancer cells. In summary, we demonstrated that andrographolide antagonized v-Src action through promotion of v-Src protein degradation. Furthermore, attenuation of the Erk1/2 signaling pathway is essential for andrographolide-mediated inhibition of v-Src transformation. Our results demonstrate that andrographolide can act as a v-Src inhibitor and reveal a novel action mechanism of andrographolide.