Genomic, transcriptomic, and metabolomic analysis of Oldenlandia corymbosa reveals the biosynthesis and mode of action of anti-cancer metabolites.

Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound.

The role of caspases as executioners of apoptosis.

Caspases are a family of cysteine aspartyl proteases mostly involved in the execution of apoptotic cell death and in regulating inflammation. This article focuses primarily on the evolutionarily conserved function of caspases in apoptosis. We summarise which caspases are involved in apoptosis, how they are activated and regulated, and what substrates they target for cleavage to orchestrate programmed cell death by apoptosis.

WBP2 promotes gastric cancer cell migration via novel targeting of LATS2 kinase in the Hippo tumor suppressor pathway.

Dysregulation of signaling pathways is responsible for many human diseases. The lack of understanding of the molecular etiology of gastric cancer (GC) poses a substantial challenge to the development of effective cancer therapy. To better understand the molecular mechanisms underlying the pathogenesis of GC, which will facilitate the identification and development of effective therapeutic approaches to improve patient outcomes, mass spectrometry-based phosphoproteomics analysis was performed to map the global molecular changes in GC. A total of 530 proteins with altered phosphorylation levels were detected across a panel of 15 normal and GC cell lines. WW domain-binding protein 2 (WBP2) was validated to be upregulated in a subset of GC cell lines. WBP2 is overexpressed in 61% cases of GC compared to non-cancer tissues and high WBP2 expression correlates with poor clinical outcomes. WBP2 was found to be required for GC cell migration but is dispensable for cell growth and proliferation. WBP2 knockdown increased p-LATS2 with a concomitant increase in p-YAP, resulting in the cytoplasmic retention of YAP and ultimately the inhibition of YAP/TEAD activity and downregulation of TEAD target genes--CTGF and CYR61. Importantly, the loss of LATS2 reversed the activation of Hippo pathway caused by WBP2 knockdown, indicating that WBP2 acts through LATS2 to exert its function on the Hippo pathway. Moreover, WBP2 interacted with LATS2 to inhibit its phosphorylation and activity. In conclusion, our study established a pivotal role for WBP2 in the promotion of GC cell migration via a novel mechanism that inactivates the Hippo pathway transducer LATS2.

Genetic localization of the orevactaene/epipyrone biosynthetic gene cluster in Epicoccum nigrum.

Epipyrone (EPN)-A (syn. orevactaene) is a polyketide compound of 3-d-galactosyl-4-hydroxy-2-pyrone with a modified heptaene acyl moiety, produced from Epicoccum nigrum and was reported to have various biological activities. Genome analysis identified a hypothetical EPN biosynthetic gene cluster (BGC) composed of the four genes epnABCD, which encode a highly-reducing fungal polyketide synthase, a glycosyltransferase, a cytochrome P450, and a transporter. The individual gene inactivation of epnABC resulted in the total loss of EPN production, while the inactivation of a nearby transcription factor-encoding gene had no effect on the production of EPN, substantiating that epnABCD is the EPN BGC. mRNA expression indicated no epnA transcription in the epnB knockout mutant and the occurrence of the bicistronic transcription of epnAB. This study defined an EPN BGC, which is the first blueprint reported for glycosylated 2-pyrone polyketide biosynthesis.

The transcriptional coactivator WBP2 primes triple-negative breast cancer cells for responses to Wnt signaling via the JNK/Jun kinase pathway.

The transcriptional coactivator WW domain-binding protein 2 (WBP2) is an emerging oncogene and serves as a node between the signaling protein Wnt and other signaling molecules and pathways, including epidermal growth factor receptor, estrogen receptor/progesterone receptor, and the Hippo pathway. The upstream regulation of WBP2 is well-studied, but its downstream activity remains unclear. Here, we elucidated WBP2's role in triple-negative breast cancer (TNBC), in which Wnt signaling is predominantly activated. Using RNAi coupled with RNA-Seq and MS analyses to identify Wnt/WBP2- and WBP2-dependent targets in MDA-MB-231 TNBC cells, we found that WBP2 is required for the expression of a core set of genes in Wnt signaling. These included AXIN2, which was essential for Wnt/WBP2-mediated breast cancer growth and migration. WBP2 also regulated a much larger set of genes and proteins independently of Wnt, revealing that WBP2 primes cells to Wnt activity by up-regulating G protein pathway suppressor 1 (GPS1) and TRAF2- and NCK-interacting kinase (TNIK). GPS1 activated the c-Jun N-terminal kinase (JNK)/Jun pathway, resulting in a positive feedback loop with TNIK that mediated Wnt-induced AXIN2 expression. WBP2 promoted TNBC growth by integrating JNK with Wnt signaling, and its expression profoundly influenced the sensitivity of TNBC to JNK/TNIK inhibitors. In conclusion, WBP2 links JNK to Wnt signaling in TNBC. GPS1 and TNIK are constituents of a WBP2-initiated cascade that primes responses to Wnt ligands and are also important for TNBC biology. We propose that WBP2 is a potential drug target for JNK/TNIK-based precision medicine for managing TNBC.

Phosphorylation of E-box binding USF-1 by PI3K/AKT enhances its transcriptional activation of the WBP2 oncogene in breast cancer cells.

WW domain binding protein 2 (WBP2), a transcriptional coactivator, plays a vital role in breast tumorigenesis. It positively regulates estrogen receptor, Hippo, and Wnt pathways, which subsequently enhance the transcription of downstream target genes contributing to cancer. Understanding the regulation of the expression and activity of WBP2 oncoprotein has implication in cancer therapy. We have previously reported that WBP2 is regulated at the post-translational and post-transcriptional levels. However, its regulation at the transcriptional level is not known. In this study, the minimal promoter region of WBP2 that is critical for its transcription was identified. The E-box motif in the WBP2 promoter was demonstrated to be essential for its transcription. The E-box binding protein upstream stimulatory factor 1 (USF-1) was discovered to be a key transcription factor for WBP2 by yeast one-hybrid analysis and was validated through reporter and chromatin immunoprecipitation assays and tandem mass spectrometry, which also suggested that USF-1 acts by regulating a network of genes, in addition to WBP2, associated with cell movement, proliferation, cell-cycle, and survival cellular processes. USF-1 is overexpressed in majority of the breast cancer cell lines and tissues tested, and has profound effects on cancer cell proliferation. USF-1-mediated transcription of WBP2 was demonstrated to be inducible by insulin, which led to AKT-mediated phosphorylation of USF-1 that modulated its ability to bind to the WBP2 promoter and activate its transcription. This study sheds new light onto the regulation of the WBP2 oncogene at the transcriptional level by a novel oncogenic transcription factor, USF-1. USF-1 is a potential drug target for treatment of WBP2-positive breast cancer.-Ramos, A., Miow, Q. H., Liang, X., Lin, Q. S., Putti, T. C., Lim, Y. P. Phosphorylation of E-box binding USF-1 by PI3K/AKT enhances its transcriptional activation of the WBP2 oncogene in breast cancer cells.

p75 neurotrophin receptor interacts with and promotes BACE1 localization in endosomes aggravating amyloidogenesis.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deposition of amyloid beta (Aß) and dysregulation of neurotrophic signaling, causing synaptic dysfunction, loss of memory, and cell death. The expression of p75 neurotrophin receptor is elevated in the brain of AD patients, suggesting its involvement in this disease. However, the exact mechanism of its action is not yet clear. Here, we show that p75 interacts with beta-site amyloid precursor protein cleaving enzyme-1 (BACE1), and this interaction is enhanced in the presence of Aß. Our results suggest that the colocalization of BACE1 and amyloid precursor protein (APP) is increased in the presence of both Aß and p75 in cortical neurons. In addition, the localization of APP and BACE1 in early endosomes is increased in the presence of Aß and p75. An increased phosphorylation of APP-Thr668 and BACE1-Ser498 by c-Jun N-terminal kinase (JNK) in the presence of Aß and p75 could be responsible for this localization. In conclusion, our study proposes a potential involvement in amyloidogenesis for p75, which may represent a future therapeutic target for AD. Cover Image for this Issue: doi. 10.1111/jnc.14163.

BDNF Val66Met in preclinical Alzheimer's disease is associated with short-term changes in episodic memory and hippocampal volume but not serum mBDNF.

BACKGROUND: The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism Met allele exacerbates amyloid (Aß) related decline in episodic memory (EM) and hippocampal volume (HV) over 36-54 months in preclinical Alzheimer's disease (AD). However, the extent to which Aß+ and BDNF Val66Met is related to circulating markers of BDNF (e.g. serum) is unknown. We aimed to determine the effect of Aß and the BDNF Val66Met polymorphism on levels of serum mBDNF, EM, and HV at baseline and over 18-months. METHODS: Non-demented older adults (n = 446) underwent Aß neuroimaging and BDNF Val66Met genotyping. EM and HV were assessed at baseline and 18 months later. Fasted blood samples were obtained from each participant at baseline and at 18-month follow-up. Aß PET neuroimaging was used to classify participants as Aß- or Aß+. RESULTS: At baseline, Aß+ adults showed worse EM impairment and lower serum mBDNF levels relative to Aß- adults. BDNF Val66Met polymorphism did not affect serum mBDNF, EM, or HV at baseline. When considered over 18-months, compared to Aß- Val homozygotes, Aß+ Val homozygotes showed significant decline in EM and HV but not serum mBDNF. Similarly, compared to Aß+ Val homozygotes, Aß+ Met carriers showed significant decline in EM and HV over 18-months but showed no change in serum mBDNF. CONCLUSION: While allelic variation in BDNF Val66Met may influence Aß+ related neurodegeneration and memory loss over the short term, this is not related to serum mBDNF. Longer follow-up intervals may be required to further determine any relationships between serum mBDNF, EM, and HV in preclinical AD.

Huntingtin-associated protein-1 is a synapsin I-binding protein regulating synaptic vesicle exocytosis and synapsin I trafficking.

Huntingtin-associated protein-1 (HAP1) is involved in intracellular trafficking, vesicle transport, and membrane receptor endocytosis. However, despite such diverse functions, the role of HAP1 in the synaptic vesicle (SV) cycle in nerve terminals remains unclear. Here, we report that HAP1 functions in SV exocytosis, controls total SV turnover and the speed of vesicle fusion in nerve terminals and regulates glutamate release in cortical brain slices. We found that HAP1 interacts with synapsin I, an abundant neuronal phosphoprotein that associates with SVs during neurotransmitter release and regulates synaptic plasticity and neuronal development. The interaction between HAP1 with synapsin I was confirmed by reciprocal co-immunoprecipitation of the endogenous proteins. Furthermore, HAP1 co-localizes with synapsin I in cortical neurons as discrete puncta. Interestingly, we find that synapsin I localization is specifically altered in Hap1(-/-) cortical neurons without an effect on the localization of other SV proteins. This effect on synapsin I localization was not because of changes in the levels of synapsin I or its phosphorylation status in Hap1(-/-) brains. Furthermore, fluorescence recovery after photobleaching in transfected neurons expressing enhanced green fluorescent protein-synapsin Ia demonstrates that loss of HAP1 protein inhibits synapsin I transport. Thus, we demonstrate that HAP1 regulates SV exocytosis and may do so through binding to synapsin I. The Proposed mechanism of synapsin I transport mediated by HAP1 in neurons. HAP1 interacts with synapsin I, regulating the trafficking of synapsin I containing vesicles and/or transport packets, possibly through its engagement of microtubule motors. The absence of HAP1 reduces synapsin I transport and neuronal exocytosis. These findings provide insights into the processes of neuronal trafficking and synaptic signaling.

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin.

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.

Proteomic profiling predicts drug response to novel targeted anticancer therapeutics.

Most recently approved anti-cancer drugs by the US FDA are targeted therapeutic agents and this represents an important trend for future anticancer therapy. Unlike conventional chemotherapy that rarely considers individual differences, it is crucial for targeted therapies to identify the beneficial subgroup of patients for the treatment. Currently, genomics and transcriptomics are the major 'omic' analytics used in studies of drug response prediction. However, proteomic profiling excels both in its advantages of directly detecting an instantaneous dynamic of the whole proteome, which contains most current diagnostic markers and therapeutic targets. Moreover, proteomic profiling improves understanding of the mechanism for drug resistance and helps finding optimal combination therapy. This article reviews the recent success of applications of proteomic analytics in predicting the response to targeted anticancer therapeutics, and discusses the potential avenues and pitfalls of proteomic platforms and techniques used most in the field.

S100P is Overexpressed in Squamous Cell and Adenosquamous Carcinoma Subtypes of Endometrial Cancer and Promotes Cancer Cell Proliferation and Invasion.

S100P is known to affect tumor development and metastasis of various cancers, but its role in endometrial cancer is unclear. We reported that S100P expression was dramatically elevated in both endometrial squamous cell carcinoma and adenosquamous carcinoma, but not in adenocarcinoma and normal endometrial samples. Moreover, we revealed an oncogenic role of S100P promoting cell proliferation, invasion, and migration while reducing apoptosis, possibly via its upregulation and/or activation of receptors of advanced glycation end products and consequently the oncogenic PI3K-AKT and MAPK pathways. Therefore, S100P might be a specific biomarker and a potential drug target for squamous cell and adenosquamous carcinoma subtypes of endometrial cancer.

In vitro secretomics study of pterygium-derived fibroblasts by iTRAQ-based quantitative proteomics strategy.

Pterygium is a triangular shaped ocular fibrous surface lesion growing from conjunctiva towards central cornea, causing ocular irritation, astigmatism, and visual disturbance. The condition is characterized by epithelial proliferation, fibrovascular growth, chronic inflammation, and prominent extracellular matrix remodeling. Studies have suggested that aberrant extracellular proteins secreted by fibroblasts lead to abnormal matrix production and tissue invasion contributing to the development of the disease. In this study, secreted proteins collected from paired pterygium and conjunctival fibroblasts in vitro were identified and quantified by LC-MS iTRAQ-based analysis, in which 433 proteins common to all samples were identified. Among these proteins, 48.0% (208) were classified as classically secreted proteins, 17.1% (74) were exported out of the cells via non-classical secretion pathways, and 31.2% (135) were exosome proteins. A minority (3.7%) was not previously known to be secreted, or might be contaminants. 31 and 27 proteins were found up- or down-regulated in the conditioned media of pterygium fibroblasts relative to the media of control cells, respectively. Molecular function analysis showed that these proteins either belonged to catalytic proteins, structural molecules or were involved with receptor activities and protein binding. Further pathway analysis revealed that these proteins were involved in ECM-receptor interaction, focal adhesion, cancer-related, p53 signaling, complement and coagulation, and TGF-beta signaling pathways. These molecules identified may serve as extracellular ligands to activate intracellular pathways, possibly serving as potential therapeutic targets.

Development of mature BDNF-specific sandwich ELISA.

Mature brain-derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme-linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF-specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti-BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross-reactivity to human recombinant NT-3, NT-4, nerve growth factor and negligible cross-reactivity (0.99-4.99%) for proBDNF compared to commercial ELISA kits (33.18-91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1)(-/-) and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity. As the function of proBDNF differs from mBDNF (mature BDNF), it is necessary to establish an ELISA specific for the detection of mBDNF. Here, we present a novel sandwich ELISA which detects mBDNF with high specificity. This new ELISA will be useful for the measurement of mBDNF levels with high specificity in various human and animal tissues. proBDNF, precursor of BDNF; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; NGF, nerve growth factor.

Divide and conquer: subproteomic approaches toward gastric cancer biomarker and drug target discovery.

The discovery of biomarkers for early detection and treatment for gastric cancer are two important gaps that proteomics have the potential to fill. Advancements in mass spectrometry, sample preparation and separation strategies are crucial to proteomics-based discoveries and subsequent translations from bench to bedside. A great number of studies exploiting various subproteomic approaches have emerged for higher-resolution analysis (compared with shotgun proteomics) that permit interrogation of different post-translational and subcellular compartmentalized forms of the same proteins as determinants of disease phenotypes. This is a unique and key strength of proteomics over genomics. In this review, the salient features, competitive edges and pitfalls of various subproteomic approaches are discussed. We also highlight valuable insights from several subproteomic studies that have increased our understanding of the molecular etiology of gastric cancer and the findings that led to the discovery of potential biomarkers/drug targets that were otherwise not revealed by conventional shotgun expression proteomics.

The use of transformed IMR90 cell model to identify the potential extra-telomeric effects of hTERT in cell migration and DNA damage response.

BACKGROUND: Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomesase, is responsible for telomere maintenance and its reactivation is implicated in almost 90% human cancers. Recent evidences show that hTERT is essential for neoplastic transformation independent of its canonical function. However, the roles of hTERT in the process remain elusive. In the current work, we explore the extra-telomeric role of hTERT in the neoplastic transformation of fibroblast IMR90. RESULTS: Here we established transformed IMR90 cells by co-expression of three oncogenic factors, namely, H-Ras, SV40 Large-T antigen and hTERT (RSH). The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities. Furthermore, the RSH-transformed cells showed enhanced migration capability which was also observed in IMR90 cells expressing hTERT alone, indicating that hTERT plays a role in cell migration, and thus possibly contribute to their metastatic potential during tumor transformation. This notion was further supported by our microarray analysis. In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR). CONCLUSIONS: Collectively, our study revealed the extra-telomeric effects of hTERT in cell migration and DDR during neoplastic transformation.

TLR7-dependent eosinophil degranulation links psoriatic skin inflammation to small intestinal inflammatory changes in mice.

Recent evidence of gut microbiota dysbiosis in the context of psoriasis and the increased cooccurrence of inflammatory bowel disease and psoriasis suggest a close relationship between skin and gut immune responses. Using a mouse model of psoriasis induced by the Toll-like receptor (TLR) 7 ligand imiquimod, we found that psoriatic dermatitis was accompanied by inflammatory changes in the small intestine associated with eosinophil degranulation, which impaired intestinal barrier integrity. Inflammatory responses in the skin and small intestine were increased in mice prone to eosinophil degranulation. Caco-2 human intestinal epithelial cells were treated with media containing eosinophil granule proteins and exhibited signs of inflammation and damage. Imiquimod-induced skin and intestinal changes were attenuated in eosinophil-deficient mice, and this attenuation was counteracted by the transfer of eosinophils. Imiquimod levels and the distribution of eosinophils were positively correlated in the intestine. TLR7-deficient mice did not exhibit intestinal eosinophil degranulation but did exhibit attenuated inflammation in the skin and small intestine following imiquimod administration. These results suggest that TLR7-dependent bidirectional skin-to-gut communication occurs in psoriatic inflammation and that inflammatory changes in the intestine can accelerate psoriasis.

Caspase-2 protects against ferroptotic cell death.

Caspase-2, one of the most evolutionarily conserved members of the caspase family, is an important regulator of the cellular response to oxidative stress. Given that ferroptosis is suppressed by antioxidant defense pathways, such as that involving selenoenzyme glutathione peroxidase 4 (GPX4), we hypothesized that caspase-2 may play a role in regulating ferroptosis. This study provides the first demonstration of an important and unprecedented function of caspase-2 in protecting cancer cells from undergoing ferroptotic cell death. Specifically, we show that depletion of caspase-2 leads to the downregulation of stress response genes including SESN2, HMOX1, SLC7A11, and sensitizes mutant-p53 cancer cells to cell death induced by various ferroptosis-inducing compounds. Importantly, the canonical catalytic activity of caspase-2 is not required for its role and suggests that caspase-2 regulates ferroptosis via non-proteolytic interaction with other proteins. Using an unbiased BioID proteomics screen, we identified novel caspase-2 interacting proteins (including heat shock proteins and co-chaperones) that regulate cellular responses to stress. Finally, we demonstrate that caspase-2 limits chaperone-mediated autophagic degradation of GPX4 to promote the survival of mutant-p53 cancer cells. In conclusion, we document a novel role for caspase-2 as a negative regulator of ferroptosis in cells with mutant p53. Our results provide evidence for a novel function of caspase-2 in cell death regulation and open potential new avenues to exploit ferroptosis in cancer therapy.

The Drosophila ZNRF1/2 homologue, detour, interacts with HOPS complex and regulates autophagy.

Autophagy, the process of elimination of cellular components by lysosomal degradation, is essential for animal development and homeostasis. Using the autophagy-dependent Drosophila larval midgut degradation model we identified an autophagy regulator, the RING domain ubiquitin ligase CG14435 (detour). Depletion of detour resulted in increased early-stage autophagic vesicles, premature tissue contraction, and overexpression of detour or mammalian homologues, ZNRF1 and ZNRF2, increased autophagic vesicle size. The ablation of ZNRF1 or ZNRF2 in mammalian cells increased basal autophagy. We identified detour interacting proteins including HOPS subunits, deep orange (dor/VPS18), Vacuolar protein sorting 16A (VPS16A), and light (lt/VPS41) and found that detour promotes their ubiquitination. The detour mutant accumulated autophagy-related proteins in young adults, displayed premature ageing, impaired motor function, and activation of innate immunity. Collectively, our findings suggest a role for detour in autophagy, likely through regulation of HOPS complex, with implications for healthy aging.

Identification of autocrine growth factors secreted by CHO cells for applications in single-cell cloning media.

Chinese hamster ovary (CHO) cell lines are widely used for the expression of therapeutic recombinant proteins, including monoclonal antibodies and other biologics. For manufacturing, cells derived from a single-cell clone are typically used to ensure product consistency. Presently, fetal bovine serum (FBS) is commonly used to support low cell density cultures to obtain clonal cell populations because cells grow slowly, or even do not survive at low cell densities in protein-free media. However, regulatory authorities have discouraged the use of FBS to reduce the risk of contamination by adventitious agents from animal-derived components. In this study, we demonstrated how a complementary mass spectrometry-based shotgun proteomics strategy enabled the identification of autocrine growth factors in CHO cell-conditioned media, which has led to the development of a fully defined single-cell cloning media that is serum and animal component-free. Out of 290 secreted proteins that were identified, eight secreted growth factors were reported for the first time from CHO cell cultures. By supplementing a combination of these growth factors to protein-free basal media, single cell growth of CHO cells was improved with cloning efficiencies of up to 30%, a 2-fold improvement compared to unsupplemented basal media. Complementary effects of these autocrine growth factors with other paracrine growth factors were also demonstrated when the mixture improved cloning efficiency to 42%, similar to that for the conditioned medium.