I am currently working on several projects to understand the control of gastrointestinal and cancer stem cell biology, especially how critical intrinsic genetic mutations and extrinsic extracellular components within the microenvironment influence cell behaviors. Stem cells of the gastrointestinal tract give rise to the surface lining of the epithelium, and must continuously produce new cells to replace those shed into the lumen throughout the lifespan. When mutations accumulate in these stem cells, they can grow uncontrollably into benign polyps or malignant tumors. In Dr. Calvin Kuo’s laboratory, I have used transgenic mice and primary human organoids as the models. Human organoids provide a robust primary culture system to recapitulate 3D structure and multilineage differentiation, which represents an underutilized method for the study of stem cell and cancer biology (Lo et al, Nature Cancer 2020). I have focused my efforts on establishing next generation CRISPR/Cas9 genome editing tools in primary human organoids, and applying this powerful system to gain insight into how different signaling pathways can contribute to gastrointestinal stem cell activity and tumorigenesis (Lo et al, Cancer Discovery 2021).
Honors & Awards
The NIH Pathway to Independence Award (K99/R00), National Institutes of Health (NIH) / National Cancer Institute (NCI) (2021-2026)
SCI Fellowship Award, Stanford Cancer Institute (SCI) (2021-2022)
AACR-AbbVie Scholar-in-Training Award, American Association for Cancer Research (2021)
The NCI Predoctoral to Postdoctoral Fellow Transition Award (K00), National Institutes of Health (NIH) / National Cancer Institute (NCI) (2017-2021)
The Claude W. Smith Fellowship Award, Baylor College of Medicine (2017)
The NCI Predoctoral to Postdoctoral Fellow Transition Award (F99), National Institutes of Health (NIH) / National Cancer Institute (NCI) (2016-2017)
The Dean’s Award of Excellence, Baylor College of Medicine (2016)
Professor John J. Trentin Scholarship Award, Baylor College of Medicine (2015)
The Claude W. Smith Fellowship Award, Baylor College of Medicine (2015)
American Gastroenterological Association Student Abstract Prize, American Gastroenterological Association (2014)
Robert and Emma Lou Cardell Foundation Fellowship, University of Cincinnati (2013)
Doctor of Philosophy, Baylor College of Medicine, Cancer and Stem Cell Biology (2017)
Master of Science, National Tsing Hua University, Taiwan, Molecular and Cellular Biology (2008)
Bachelor of Science, China Medical University, Taiwan, Biological Science and Technology (2006)
A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation.
Mutations in ARID1A rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation. Genetic Wnt/B-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells.
View details for DOI 10.1158/2159-8290.CD-20-1109
View details for PubMedID 33451982
CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.
2020; 580 (7801): 136-141
Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.
View details for DOI 10.1038/s41586-020-2099-x
View details for PubMedID 32238925
Applications of organoids for cancer biology and precision medicine.
2020; 1: 761–773
View details for DOI 10.1038/s43018-020-0102-y
SPDEF Induces Quiescence of Colorectal Cancer Cells by Changing the Transcriptional Targets of ß-catenin.
The canonical Wnt signaling pathway activates the transcriptional activity of β-catenin. This pathway is often activated in colorectal cancer cells, but strategies to block it in tumors have not been effective. The SAM pointed domain containing ETS transcription factor (SPDEF) suppresses formation of colon tumors by unclear mechanisms. We investigated these mechanisms and the effects of SPDEF on β-catenin activity in mouse models of colorectal cancer (CRC), CRC cell lines, and mouse and human normal and cancer colonoids.We performed studies of Lgr5(CreERT2); β-catenin(exon3); Rosa26(LSL-rtta-ires-EGFP); TRE-Spdef mice, which express an oncogenic form of β-catenin in Lgr5-positive ISCs upon administration of tamoxifen and SPDEF upon administration of tetracycline. CRC lines (HCT116 and SW480) were engineered to express inducible tagged SPDEF or vector (control) and subcutaneously injected into immunodeficient NSG mice. We generated SPDEF-inducible human colonoids, including a line derived from normal rectal mucosa (control) and an adenocarcinoma line derived from a patient with germline MUTYH mutation. Full-length and truncated forms of SPDEF were expressed in CRC cells; cells were assayed for β-catenin activity and studied in immunoprecipitation and chromatin immunoprecipitation assays.Expression of SPDEF was sufficient to inhibit intestinal tumorigenesis by activated β-catenin, block tumor cell proliferation, and restrict growth of established tumors. In tumor cells with activated β -catenin, expression of SPDEF induced a quiescent state, which was reversed when SPDEF expression was stopped. In mouse and human normal and tumor-derived enteroids/colonoids, those that expressed SPDEF for 3 days were significantly smaller. SPDEF inhibited the transcriptional activity of β-catenin via a protein-protein interaction, independent of SPDEF DNA binding capacity. SPDEF disrupted β-catenin binding to TCF1 and TCF3, displacing β-catenin from enhancer regions of genes that regulate the cell cycle but not genes that regulate stem cell activities.In studies of mice and human CRC, we found that SPDEF induces a quiescent state in CRC cells by disrupting binding of β-catenin to TCF1 and TCF3 and regulation of genes that control the cell cycle. In this model, β-catenin activity determines the proliferation or quiescence of CRC cells based on the absence or presence of SPDEF.
View details for DOI 10.1053/j.gastro.2017.03.048
View details for PubMedID 28390865
Transcriptional Regulation by ATOH1 and its Target SPDEF in the Intestine.
Cellular and molecular gastroenterology and hepatology
2017; 3 (1): 51-71
The transcription factor atonal homolog 1 (ATOH1) controls the fate of intestinal progenitors downstream of the Notch signaling pathway. Intestinal progenitors that escape Notch activation express high levels of ATOH1 and commit to a secretory lineage fate, implicating ATOH1 as a gatekeeper for differentiation of intestinal epithelial cells. Although some transcription factors downstream of ATOH1, such as SPDEF, have been identified to specify differentiation and maturation of specific cell types, the bona fide transcriptional targets of ATOH1 still largely are unknown. Here, we aimed to identify ATOH1 targets and to identify transcription factors that are likely to co-regulate gene expression with ATOH1.We used a combination of chromatin immunoprecipitation and messenger RNA-based high-throughput sequencing (ChIP-seq and RNA-seq), together with cell sorting and transgenic mice, to identify direct targets of ATOH1, and establish the epistatic relationship between ATOH1 and SPDEF.By using unbiased genome-wide approaches, we identified more than 700 genes as ATOH1 transcriptional targets in adult small intestine and colon. Ontology analysis indicated that ATOH1 directly regulates genes involved in specification and function of secretory cells. De novo motif analysis of ATOH1 targets identified SPDEF as a putative transcriptional co-regulator of ATOH1. Functional epistasis experiments in transgenic mice show that SPDEF amplifies ATOH1-dependent transcription but cannot independently initiate transcription of ATOH1 target genes.This study unveils the direct targets of ATOH1 in the adult intestines and illuminates the transcriptional events that initiate the specification and function of intestinal secretory lineages.
View details for DOI 10.1016/j.jcmgh.2016.10.001
View details for PubMedID 28174757
Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration
2015; 528 (7583): 560-?
Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch and epidermal growth factor (EGF) signals supporting Lgr5(+) crypt base columnar ISCs for normal epithelial maintenance. However, little is known about the regulation of the ISC compartment after tissue damage. Using ex vivo organoid cultures, here we show that innate lymphoid cells (ILCs), potent producers of interleukin-22 (IL-22) after intestinal injury, increase the growth of mouse small intestine organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both mouse and human intestinal organoids, increasing proliferation and promoting ISC expansion. IL-22 induced STAT3 phosphorylation in Lgr5(+) ISCs, and STAT3 was crucial for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after mouse allogeneic bone marrow transplantation enhanced the recovery of ISCs, increased epithelial regeneration and reduced intestinal pathology and mortality from graft-versus-host disease. ATOH1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independently of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support the intestinal epithelium, activating ISCs to promote regeneration.
View details for DOI 10.1038/nature16460
View details for PubMedID 26649819
SPDEF Functions as a Colorectal Tumor Suppressor by Inhibiting beta-Catenin Activity
2013; 144 (5): 1012-?
Expression of the SAM pointed domain containing ETS transcription factor (SPDEF or prostate-derived ETS factor) is regulated by Atoh1 and is required for the differentiation of goblet and Paneth cells. SPDEF has been reported to suppress the development of breast, prostate, and colon tumors. We analyzed levels of SPDEF in colorectal tumor samples from patients and its tumor-suppressive functions in mouse models of colorectal cancer (CRC).We analyzed levels of SPDEF messenger RNA and protein in more than 500 human CRC samples and more than 80 nontumor controls. Spdef(-/-)and wild-type mice (controls) were either bred with Apc(Min/+) mice, or given azoxymethane (AOM) and dextran sodium sulfate (DSS), or 1,2-dimethylhydrazine and DSS, to induce colorectal tumors. Expression of Spdef also was induced transiently by administration of tetracycline to Spdef(dox-intestine) mice with established tumors, induced by the combination of AOM and DSS or by breeding with Apc(Min/+) mice. Colon tissues were collected and analyzed for tumor number, size, grade, and for cell proliferation and apoptosis. We also analyzed the effects of SPDEF expression in HCT116 and SW480 human CRC cells.In colorectal tumors from patients, loss of SPDEF was observed in approximately 85% of tumors and correlated with progression from normal tissue, to adenoma, to adenocarcinoma. Spdef(-/-); Apc(Min/+) mice developed approximately 3-fold more colon tumors than Spdef(+/+); Apc(Min/+) mice. Likewise, Spdef(-/-) mice developed approximately 3-fold more colon tumors than Spdef(+/+) mice after administration of AOM and DSS. After administration of 1,2-dimethylhydrazine and DSS, invasive carcinomas were observed exclusively in Spdef(-/-) mice. Conversely, expression of SPDEF was sufficient to promote cell-cycle exit in cells of established adenomas from Spdef(dox-intestine); Apc(Min/+) mice and in Spdef(dox-intestine) mice after administration of AOM + DSS. SPDEF inhibited the expression of β-catenin-target genes in mouse colon tumors, and interacted with β-catenin to block its transcriptional activity in CRC cell lines, resulting in lower levels of cyclin D1 and c-MYC.SPDEF is a colon tumor suppressor and a candidate therapeutic target for colon adenomas and adenocarcinoma.
View details for DOI 10.1053/j.gastro.2013.01.043
View details for Web of Science ID 000317813900030
View details for PubMedID 23376423
View details for PubMedCentralID PMC3738069
T cell-derived interferon-γ programs stem cell death in immune-mediated intestinal damage.
2019; 4 (42)
Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.
View details for DOI 10.1126/sciimmunol.aay8556
View details for PubMedID 31811055
Growth Factor Independent 1 is a tumor suppressor gene in colorectal cancer.
Molecular cancer research : MCR
Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death in the United States. Growth Factor Independent 1 (GFI1) is a zinc finger transcriptional repressor responsible for controlling secretory cell differentiation in the small intestine and colon. GFI1 plays a significant role in the development of human malignancies, including leukemia, lung cancer and prostate cancer. However, the role of GFI1 in CRC progression is largely unknown. Our results demonstrate that RNA and protein expression of GFI1 are reduced in advanced stage non-mucinous CRC. Subcutaneous tumor xenograft models demonstrated that the re-expression of GFI1 in 4 different human CRC cell lines inhibits tumor growth. To further investigate the role of Gfi1 in de novo colorectal tumorigenesis, we developed transgenic mice harboring a deletion of Gfi1 in the distal colon driven by CDX2-cre (Gfi1F/F; CDX2-cre) and crossed them with ApcMin/+ mice (ApcMin/+; Gfi1F/F; CDX2-cre). Loss of Gfi1 significantly increased the total number of colorectal adenomas compared to littermate controls with an APC mutation alone. Furthermore, we found that compound (ApcMin/+; Gfi1F/F; CDX2-cre) mice develop larger adenomas, invasive carcinoma, as well as hyperplastic lesions expressing the neuroendocrine marker chromogranin A, a feature that has not been previously described in APC-mutant tumors in mice. Collectively, these results demonstrate that GFI1 acts as a tumor suppressor gene in colorectal cancer, where deficiency of Gfi1 promotes malignancy in the colon. Implications: These findings reveal that GFI1 functions as a tumor suppressor gene in colorectal tumorigenesis.
View details for DOI 10.1158/1541-7786.MCR-18-0666
View details for PubMedID 30606770
The Intestinal Stem Cell Niche: Homeostasis and Adaptations.
Trends in cell biology
The intestinal epithelium is a rapidly renewing cellular compartment. This constant regeneration is a hallmark of intestinal homeostasis and requires a tightly regulated balance between intestinal stem cell (ISC) proliferation and differentiation. Since intestinal epithelial cells directly contact pathogenic environmental factors that continuously challenge their integrity, ISCs must also actively divide to facilitate regeneration and repair. Understanding niche adaptations that maintain ISC activity during homeostatic renewal and injury-induced intestinal regeneration is therefore a major and ongoing focus for stem cell biology. Here, we review recent concepts and propose an active interconversion of the ISC niche between homeostasis and injury-adaptive states that is superimposed upon an equally dynamic equilibrium between active and reserve ISC populations.
View details for PubMedID 30195922
SOX4 Promotes ATOH1-independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates.
The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce post-mitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the; transcription factor ATOH1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type 2 immune; responses to parasite infections and appear to differentiate independently of ATOH1. We; investigated the role of SOX4 in ISC differentiation METHODS: We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre;SOX4cKO) and mice without disruption of Sox4 (control mice). Crypt- and single cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative PCR, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 (IL13) and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice), and single-cell RNA-seq analysis, were used to identify cells that co-express Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1iKO) mice and assessed differentiation.SOX4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly following helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by IL13 in control organoids; SOX4cKO knockout mice had impaired tuft cell hyperplasia and parasite clearance following infection with helminths. In scRNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISCs, progenitors, and tuft cells; 12.5% of Sox4-; expressing cells co-expressed Atoh1 and were enriched for enteroendocrine cells. In; organoids, overexpression of SOX4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of ATOH1.We found SOX4 to promote tuft and enteroendocrine cell lineage allocation independently of ATOH1. These results challenge the long-standing model in which ATOH1 is the only regulator of secretory differentiation in the intestine, and are relevant for understanding epithelial responses to parasitic infection.
View details for DOI 10.1053/j.gastro.2018.07.023
View details for PubMedID 30055169
Epithelial WNT Ligands Are Essential Drivers of Intestinal Stem Cell Activation.
2018; 22 (4): 1003-1015
Intestinal stem cells (ISCs) maintain and repair the intestinal epithelium. While regeneration after ISC-targeted damage is increasingly understood, injury-repair mechanisms that direct regeneration following injuries to differentiated cells remain uncharacterized. The enteric pathogen, rotavirus, infects and damages differentiated cells while sparing all ISC populations, thus allowing the unique examination of the response of intact ISC compartments during injury-repair. Upon rotavirus infection in mice, ISC compartments robustly expand and proliferating cells rapidly migrate. Infection results specifically in stimulation of the active crypt-based columnar ISCs, but not alternative reserve ISC populations, as is observed after ISC-targeted damage. Conditional ablation of epithelial WNT secretion diminishes crypt expansion and ISC activation, demonstrating a previously unknown function of epithelial-secreted WNT during injury-repair. These findings indicate a hierarchical preference of crypt-based columnar cells (CBCs) over other potential ISC populations during epithelial restitution and the importance of epithelial-derived signals in regulating ISC behavior.
View details for DOI 10.1016/j.celrep.2017.12.093
View details for PubMedID 29386123
The ErbB3 receptor tyrosine kinase negatively regulates Paneth cells by PI3K-dependent suppression of Atoh1
CELL DEATH AND DIFFERENTIATION
2017; 24 (5): 855-865
Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. PC dysfunction is associated with disorders such as Crohn's disease and necrotizing enterocolitis, but the specific pathways regulating PC development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of PC differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of PCs as early as postnatal day 7, and substantially increased the number of mature PCs in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1(+) cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the PC marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Expansion of the PC compartment in ErbB3-null intestines was accompanied with elevated ER stress and inflammation markers, raising the possibility that negative regulation of PCs by ErbB3 is necessary to maintain homeostasis. Taken together, our data suggest that ErbB3 restricts PC numbers through PI3K-mediated suppression of Atoh1 levels leading to inhibition of PC differentiation, with important implications for regulation of the ISC niche.
View details for DOI 10.1038/cdd.2017.27
View details for Web of Science ID 000400655600010
View details for PubMedID 28304405
View details for PubMedCentralID PMC5423110
Activated STAT5 confers resistance to intestinal injury by increasing intestinal stem cell proliferation and regeneration.
Stem cell reports
2015; 4 (2): 209-225
Intestinal epithelial stem cells (IESCs) control the intestinal homeostatic response to inflammation and regeneration. The underlying mechanisms are unclear. Cytokine-STAT5 signaling regulates intestinal epithelial homeostasis and responses to injury. We link STAT5 signaling to IESC replenishment upon injury by depletion or activation of Stat5 transcription factor. We found that depletion of Stat5 led to deregulation of IESC marker expression and decreased LGR5(+) IESC proliferation. STAT5-deficient mice exhibited worse intestinal histology and impaired crypt regeneration after γ-irradiation. We generated a transgenic mouse model with inducible expression of constitutively active Stat5. In contrast to Stat5 depletion, activation of STAT5 increased IESC proliferation, accelerated crypt regeneration, and conferred resistance to intestinal injury. Furthermore, ectopic activation of STAT5 in mouse or human stem cells promoted LGR5(+) IESC self-renewal. Accordingly, STAT5 promotes IESC proliferation and regeneration to mitigate intestinal inflammation. STAT5 is a functional therapeutic target to improve the IESC regenerative response to gut injury.
View details for DOI 10.1016/j.stemcr.2014.12.004
View details for PubMedID 25579133
View details for PubMedCentralID PMC4325270
- Biology of intestinal epithelial stem cells Intestinal Tumorigenesis Springer. 2015: 55–99
The Ron receptor tyrosine kinase activates c-Abl to promote cell proliferation through tyrosine phosphorylation of PCNA in breast cancer
2014; 33 (11): 1429-1437
Multiple growth pathways lead to enhanced proliferation in malignant cells. However, how the core machinery of DNA replication is regulated by growth signaling remains largely unclear. The sliding clamp proliferating cell nuclear antigen (PCNA) is an indispensable component of the DNA machinery responsible for replicating the genome and maintaining genomic integrity. We previously reported that epidermal growth factor receptor (EGFR) triggered tyrosine 211 (Y211) phosphorylation of PCNA, which in turn stabilized PCNA on chromatin to promote cell proliferation. Here we show that the phosphorylation can also be catalyzed by the non-receptor tyrosine kinase c-Abl. We further demonstrate that, in the absence of EGFR, signaling to PCNA can be attained through the activation of the Ron receptor tyrosine kinase and the downstream non-receptor tyrosine kinase c-Abl. We show that Ron and c-Abl form a complex, and that activation of Ron by its ligand, hepatocyte growth factor-like protein (HGFL), stimulates c-Abl kinase activity, which in turn directly phosphorylates PCNA at Y211 and leads to an increased level of chromatin-associated PCNA. Correspondingly, HGFL-induced Ron activation resulted in Y211 phosphorylation of PCNA while silencing of c-Abl blocked this effect. We show that c-Abl and Y211 phosphorylation of PCNA is an important axis downstream of Ron, which is required for cell proliferation. Treatment with a specific peptide that inhibits Y211 phosphorylation of PCNA or with the c-Abl pharmacological inhibitor imatinib suppressed HGFL-induced cell proliferation. Our findings identify the pathway of Ron-c-Abl-PCNA as a mechanism of oncogene-induced cell proliferation, with potentially important implications for development of combination therapy of breast cancer.
View details for DOI 10.1038/onc.2013.84
View details for Web of Science ID 000332943500010
View details for PubMedID 23542172
View details for PubMedCentralID PMC4064789
Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2013; 430 (1): 43-48
Clonal proliferation is an obligatory component of adipogenesis. Although several cell cycle regulators are known to participate in the transition between pre-adipocyte proliferation and terminal adipocyte differentiation, how the core DNA synthesis machinery is coordinately regulated in adipogenesis remains elusive. PCNA (Proliferating Cell Nuclear Antigen) is an indispensable component for DNA synthesis during proliferation. Here we show that PCNA is subject to phosphorylation at the highly conserved tyrosine residue 114 (Y114). Replacing the Y114 residue with phenylalanine (Y114F), which is structurally similar to tyrosine but cannot be phosphorylated, does not affect normal animal development. However, when challenged with high fat diet, mice carrying homozygous Y114F alleles (PCNA(F/F)) are resistant to adipose tissue enlargement in comparison to wild-type (WT) mice. Mouse embryonic fibroblasts (MEFs) harboring WT or Y114F mutant PCNA proliferate at similar rates. However, when subjected to adipogenesis induction in culture, PCNA(F/F) MEFs are not able to re-enter the cell cycle and fail to form mature adipocytes, while WT MEFs undergo mitotic clonal expansion in response to the adipogenic stimulation, accompanied by enhanced Y114 phosphorylation of PCNA, and differentiate to mature adipocytes. Consistent with the function of Y114 phosphorylation in clonal proliferation in adipogenesis, fat tissues isolated from WT mice contain significantly more adipocytes than those isolated from PCNA(F/F) mice. This study identifies a critical role for PCNA in adipose tissue development, and for the first time identifies a role of the core DNA replication machinery at the interface between proliferation and differentiation.
View details for DOI 10.1016/j.bbrc.2012.11.047
View details for Web of Science ID 000314320700008
View details for PubMedID 23201573
View details for PubMedCentralID PMC3724334
Epidermal Growth Factor Receptor Protects Proliferating Cell Nuclear Antigen from Cullin 4A Protein-mediated Proteolysis
JOURNAL OF BIOLOGICAL CHEMISTRY
2012; 287 (32): 27148-27157
Proliferating cell nuclear antigen (PCNA) is an essential component for DNA synthesis upon growth stimulation. It has been shown that phosphorylation of PCNA at Tyr-211 by the EGF receptor (EGFR) protects PCNA from polyubiquitylation and degradation, whereas blocking phosphorylation induces ubiquitylation-mediated degradation of the chromatin-bound, but not the -unbound, PCNA, and suppresses cell proliferation. However, the ubiquitin E3 ligase linking growth signaling to the proteolysis of PCNA and the underlying regulatory mechanism remain to be identified. Here we show that, in the absence of Tyr-211 phosphorylation, PCNA is subject to polyubiquitylation at Lys-164 by the CUL4A E3 ligase, resulting in the degradation of PCNA. Mutation of Lys-164 to arginine prevents PCNA ubiquitylation and rescues the degradation of the K164R/Y211F PCNA double mutant. Activation of EGFR inhibits the interaction of PCNA with CUL4A, whereas inhibition of EGFR leads to increased CUL4A-PCNA interaction and CUL4A-dependent ubiquitin-mediated degradation of PCNA. Substitution of endogenous PCNA with the Y211F mutant PCNA conveys enhanced sensitization to EGFR inhibition. Our findings identify CUL4A as the ubiquitin ligase linking the down-regulation of cell surface receptor tyrosine kinase to the nuclear DNA replication machinery in cancer cells.
View details for DOI 10.1074/jbc.M112.388843
View details for Web of Science ID 000307386000067
View details for PubMedID 22692198
View details for PubMedCentralID PMC3411057
Interaction of Proliferation Cell Nuclear Antigen (PCNA) with c-Abl in Cell Proliferation and Response to DNA Damages in Breast Cancer
2012; 7 (1)
Cell proliferation in primary and metastatic tumors is a fundamental characteristic of advanced breast cancer. Further understanding of the mechanism underlying enhanced cell growth will be important in identifying novel prognostic markers and therapeutic targets. Here we demonstrated that tyrosine phosphorylation of the proliferating cell nuclear antigen (PCNA) is a critical event in growth regulation of breast cancer cells. We found that phosphorylation of PCNA at tyrosine 211 (Y211) enhanced its association with the non-receptor tyrosine kinase c-Abl. We further demonstrated that c-Abl facilitates chromatin association of PCNA and is required for nuclear foci formation of PCNA in cells stressed by DNA damage as well as in unperturbed cells. Targeting Y211 phosphorylation of PCNA with a cell-permeable peptide inhibited the phosphorylation and reduced the PCNA-Abl interaction. These results show that PCNA signal transduction has an important impact on the growth regulation of breast cancer cells.
View details for DOI 10.1371/journal.pone.0029416
View details for Web of Science ID 000301070200027
View details for PubMedID 22238610
View details for PubMedCentralID PMC3251568
Overcoming Resistance to Fulvestrant (ICI182,780) by Downregulating the c-ABL Proto-Oncogene in Breast Cancer
2011; 50 (5): 383-389
Inhibiting estrogen receptor (ER) function with specific estrogen receptor modulators (SERM) can achieve a primary response in cancer patients; however, intrinsic or subsequently acquired resistance to the therapy remains a major obstacle in treatment. The pure anti-estrogen fulvestrant has been shown to be a promising antagonist of ERα in treating advanced breast cancer. However, our knowledge of the mechanisms governing cellular responsiveness to this agent is limited. Here we show that down-regulation of the nonreceptor tyrosine kinase c-ABL enhanced sensitization to fulvestrant in breast cancer cells. Blocking c-ABL kinase activity with the inhibitor imatinib further increased ERα downregulation induced by fulvestrant, decreased the number of proliferating cells entering the cell cycle, and increased cellular sensitivity to fulvestrant treatment. Conversely, introducing kinase-activated c-ABL can rescue fulvestrant-induced ERα downregulation. Consistent with the effects of imatinib, the silencing of endogenous c-ABL increased the sensitivity of breast cancer cells to fulvestrant treatment. These results demonstrate a role for c-ABL in mediating resistance to the pure anti-estrogen fulvestrant.
View details for Web of Science ID 000289414600007
View details for PubMedID 21480391
Inhibition of c-ABL Sensitizes Breast Cancer Cells to the Dual ErbB Receptor Tyrosine Kinase Inhibitor Lapatinib (GW572016)
2011; 31 (3): 789-795
The dual kinase inhibitor lapatinib (Tykerb) has been applied for advanced breast cancer. However, the effectiveness in the clinic has been elusive and the development of novel approaches to enhance the responsiveness is needed. In this study, we test whether the non-receptor tyrosine kinase c-Abl regulates the responsiveness of breast cancer cells to lapatinib and, if so, whether the combination treatment with lapatinib plus the c-ABL kinase inhibitor imatinib (STI571; Gleevec) can sensitize breast cancer cells to the treatment.The endogenous c-ABL kinase was silenced by RNA interference or inhibited by imatinib to test whether the co-treatment improves the responsiveness of the lapatinib-resistant breast cancer cell lines MDA-MB-468 and T47D, by measuring cell growth and cell-cycle progression.The responsiveness to lapatinib can be improved by targeting the function of c-ABL, suggesting that combination treatment of lapatinib plus imatinib can lead to significant gains in therapeutic benefit.
View details for Web of Science ID 000289612800007
View details for PubMedID 21498698
Targeting Tyrosine Phosphorylation of PCNA Inhibits Prostate Cancer Growth
MOLECULAR CANCER THERAPEUTICS
2011; 10 (1): 29-36
The proliferation cell nuclear antigen (PCNA) is a critical protein required for DNA replication in proliferating cells including cancer cells. However, direct inhibition of PCNA in cancer cells has been difficult due to the lack of targetable sites. We previously reported that phosphorylation of tyrosine 211 (Y211) on PCNA is important for the proliferative function of PCNA when this protein is associated with the chromatin in cancer cells. Here, we show that the Y211 phosphorylation of PCNA is a frequent event in advanced prostate cancer. To explore the potential of this signaling event in inhibition of cancer cell growth, we used a synthetic peptide, the Y211F peptide, which when present inhibits phosphorylation of Y211 on endogenous PCNA. Treatment with this peptide, but not a scrambled control peptide, resulted in S-phase arrest, inhibition of DNA synthesis, and enhanced cell death in a panel of human prostate cancer cell lines. In addition, treatment with the Y211F peptide led to decreased tumor growth in PC3-derived xenograft tumors in vivo in nude mice. Our study shows for the first time that PCNA phosphorylation at Y211 is a frequent and biologically important signaling event in prostate cancer. This study also shows a proof of concept that Y211 phosphorylation of PCNA may be used as a therapeutic target in prostate cancer cells, including cells of advanced cancers that are refractory to standard hormonal therapies.
View details for DOI 10.1158/1535-7163.MCT-10-0778
View details for Web of Science ID 000286046400004
View details for PubMedID 21220489
View details for PubMedCentralID PMC3066081