Current Role at Stanford
The main project of the Li lab is to elucidate the signaling pathways responsible for maintaining and initiating brain tumor growth. Previously the Li lab has identified an interesting protein, known as casein kinase 2, which plays an integral role in adult brain tumor growth. My goal is to expand on this initial finding and determine if casein kinase 2 could be a therapeutically relevant drug target in adult brain tumors and whether this protein plays a role in pediatric brain tumors. The role of the candidate would be the lead researcher on these projects, as well as managing and leading the undergraduate and medical students in the lab. In addition, I am responsible for maintaining the laboratory and assisting in grant writing.
Honors & Awards
Benjamin F. Graham Jr. Scholarship, Grinnell College (2000 to 2001)
Ruth L. Kirschstein National Research Service Award, NIH (2002 to 2007)
Basic Research Fellowship, ABTA (8-2008 to 8-2010)
Travel Fellowship School of Medicine Travel Grant, Stanford University School of Medicine (2010)
Casein kinase 2a regulates glioblastoma brain tumor-initiating cell growth through the ß-catenin pathway.
2015; 34 (28): 3688-3699
Glioblastoma (GBM) is the most common and fatal primary brain tumor in humans, and it is essential that new and better therapies are developed to treat this disease. Previous research suggests that casein kinase 2 (CK2) may be a promising therapeutic target for GBMs. CK2 has enhanced expression or activity in numerous cancers, including GBM, and it has been demonstrated that inhibitors of CK2 regressed tumor growth in GBM xenograft mouse models. Our studies demonstrate that the CK2 subunit, CK2α, is overexpressed in and has an important role in regulating brain tumor-initiating cells (BTIC) in GBM. Initial studies showed that two GBM cell lines (U87-MG and U138) transduced with CK2α had enhanced proliferation and anchorage-independent growth. Inhibition of CKα using siRNA or small-molecule inhibitors (TBBz, CX-4945) reduced cell growth, decreased tumor size, and increased survival rates in GBM xenograft mouse models. We also verified that inhibition of CK2α decreased the activity of a well-known GBM-initiating cell regulator, β-catenin. Loss of CK2α decreased two β-catenin-regulated genes that are involved in GBM-initiating cell growth, OCT4 and NANOG. To determine the importance of CK2α in GBM stem cell maintenance, we reduced CK2α activity in primary GBM samples and tumor spheres derived from GBM patients. We discovered that loss of CK2α activity reduced the sphere-forming capacity of BTIC and decreased numerous GBM stem cell markers, including CD133, CD90, CD49f and A2B5. Our study suggests that CK2α is involved in GBM tumorigenesis by maintaining BTIC through the regulation of β-catenin.Oncogene advance online publication, 22 September 2014; doi:10.1038/onc.2014.299.
View details for DOI 10.1038/onc.2014.299
View details for PubMedID 25241897
?-Glutamyl transferase 7 is a novel regulator of glioblastoma growth.
2015; 15: 225-?
Glioblastoma (GBM) is the most malignant primary brain tumor in adults, with a median survival time of one and a half years. Traditional treatments, including radiation, chemotherapy, and surgery, are not curative, making it imperative to find more effective treatments for this lethal disease. γ-Glutamyl transferase (GGT) is a family of enzymes that was shown to control crucial redox-sensitive functions and to regulate the balance between proliferation and apoptosis. GGT7 is a novel GGT family member that is highly expressed in brain and was previously shown to have decreased expression in gliomas. Since other members of the GGT family were found to be altered in a variety of cancers, we hypothesized that GGT7 could regulate GBM growth and formation.To determine if GGT7 is involved in GBM tumorigenesis, we modulated GGT7 expression in two GBM cell lines (U87-MG and U138) and monitored changes in tumorigenicity in vitro and in vivo.We demonstrated for the first time that GBM patients with low GGT7 expression had a worse prognosis and that 87% (7/8) of primary GBM tissue samples showed a 2-fold decrease in GGT7 expression compared to normal brain samples. Exogenous expression of GGT7 resulted in a 2- to 3-fold reduction in proliferation and anchorage-independent growth under minimal growth conditions (1% serum). Decreasing GGT7 expression using either short interfering RNA or short hairpin RNA consistently increased proliferation 1.5- to 2-fold. In addition, intracranial injections of U87-MG cells with reduced GGT7 expression increased tumor growth in mice approximately 2-fold, and decreased mouse survival. To elucidate the mechanism by which GGT7 regulates GBM growth, we analyzed reactive oxygen species (ROS) levels in GBM cells with modulated GGT7 expression. We found that enhanced GGT7 expression reduced ROS levels by 11-33%.Our study demonstrates that GGT7 is a novel player in GBM growth and that GGT7 can play a critical role in tumorigenesis by regulating anti-oxidative damage. Loss of GGT7 may increase the cellular ROS levels, inducing GBM occurrence and growth. Our findings suggest that GGT7 can be a promising biomarker and a potential therapeutic target for GBM.
View details for DOI 10.1186/s12885-015-1232-y
View details for PubMedID 25884624
View details for PubMedCentralID PMC4393868
Casein Kinase 2: a novel player in glioblastoma therapy and cancer stem cells.
Journal of molecular and genetic medicine : an international journal of biomedical research
2013; 8 (1)
Casein kinase 2 (CK2) is an oncogenic protein kinase which contributes to tumor development, proliferation, and suppression of apoptosis in multiple cancer types. The mechanism by which CK2 expression and activity leads to tumorigenesis in glioblastoma (GBM), a stage IV primary brain tumor, is being studied. Recent studies demonstrate that CK2 plays an important role in GBM formation and growth through the inhibition of tumor suppressors and activation of oncogenes. In addition, intriguing new reports indicate that CK2 may regulate GBM formation in a novel manner; CK2 may play a critical role in cancer stem cell (CSC) maintenance. Since glial CSCs have the ability to self-renew and initiate tumor growth, new treatments which target these CSCs are needed to treat this fatal disease. Inhibition of CK2 is potentially a novel method to inhibit GBM growth and reoccurrence by targeting the glial CSCs. A new, orally available, selective CK2 inhibitor, CX-4945 has had promising results when tested in cancer cell lines, in vivo xenograft models, and human clinical trials. The development of CK2 targeted inhibitors, starting with CX-4945, may lead to a new class of more effective cancer therapies.
View details for PubMedID 25264454
- The invasive nature of glioblastoma. World neurosurgery 2013; 80 (3-4): 279-280
Expression of epidermal growth factor variant III (EGFRvIII) in pediatric diffuse intrinsic pontine gliomas
JOURNAL OF NEURO-ONCOLOGY
2012; 108 (3): 395-402
Despite numerous clinical trials over the past 2 decades, the overall survival for children diagnosed with diffuse intrinsic pontine glioma (DIPG) remains 9-10 months. Radiation therapy is the only treatment with proven effect and novel therapies are needed. Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the epidermal growth factor receptor and is expressed in many tumor types but is rarely found in normal tissue. A peptide vaccine targeting EGFRvIII is currently undergoing investigation in phase 3 clinical trials for the treatment of newly diagnosed glioblastoma (GBM), the tumor in which this variant receptor was first discovered. In this study, we evaluated EGFRvIII expression in pediatric DIPG samples using immunohistochemistry with a double affinity purified antibody raised against the EGFRvIII peptide. Staining of pediatric DIPG histological samples revealed expression in 4 of 9 cases and the pattern of staining was consistent with what has been seen in EGFRvIII transfected cells as well as GBMs from adult trials. In addition, analysis of tumor samples collected immediately post mortem and of DIPG cells in culture by RT-PCR, western blot analysis, and flow cytometry confirmed EGFRvIII expression. We were therefore able to detect EGFRvIII expression in 6 of 11 DIPG cases. These data suggest that EGFRvIII warrants investigation as a target for these deadly pediatric tumors.
View details for DOI 10.1007/s11060-012-0842-3
View details for Web of Science ID 000305123800007
View details for PubMedID 22382786
View details for PubMedCentralID PMC3368992
Epidermal Growth Factor Receptor Variant III Contributes to Cancer Stem Cell Phenotypes in Invasive Breast Carcinoma
2012; 72 (10): 2657-2671
EGFRvIII is a tumor-specific variant of the epidermal growth factor receptor (EGFR). Although EGFRvIII is most commonly found in glioblastoma, its expression in other tumor types remains controversial. In this study, we investigated EGFRvIII expression and amplification in primary breast carcinoma. Our analyses confirmed the presence of EGFRvIII, but in the absence of amplification or rearrangement of the EGFR locus. Nested reverse transcriptase PCR and flow cytometry were used to detect a higher percentage of positive cases. EGFRvIII-positive cells showed increased expression of genes associated with self-renewal and epithelial-mesenchymal transition along with a higher percentage of stem-like cells. EGFRvIII also increased in vitro sphere formation and in vivo tumor formation. Mechanistically, EGFRvIII mediated its effects through the Wnt/β-catenin pathway, leading to increased β-catenin target gene expression. Inhibition of this pathway reversed the observed effects on cancer stem cell (CSC) phenotypes. Together, our findings show that EGFRvIII is expressed in primary breast tumors and contributes to CSC phenotypes in breast cancer cell lines through the Wnt pathway. These data suggest a novel function for EGFRvIII in breast tumorigenesis.
View details for DOI 10.1158/0008-5472.CAN-11-2656
View details for Web of Science ID 000307346800020
View details for PubMedID 22419663
Pathology: Commonly Monitored Glioblastoma Markers: EFGR, EGFRvIII, PTEN, and MGMT
NEUROSURGERY CLINICS OF NORTH AMERICA
2012; 23 (2): 237-?
The purpose of this article is to update the neurosurgical field on current molecular markers important to glioblastoma biology, treatment, and prognosis. The highlighted biologic markers in this article include epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and O6-methylguanine-DNA methyltransferase (MGMT).
View details for DOI 10.1016/j.nec.2012.01.011
View details for Web of Science ID 000303282100006
View details for PubMedID 22440867
The role of the c-Jun N-terminal kinase 2-alpha-isoform in non-small cell lung carcinoma tumorigenesis
2011; 30 (2): 234-244
The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2α, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2α isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2α mRNA expression than normal lung control tissue. To determine the importance of JNK2α in NSCLC progression, we reduced JNK2α expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2α had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2α induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2α can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2α2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2α is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2α.
View details for DOI 10.1038/onc.2010.414
View details for Web of Science ID 000286438900011
View details for PubMedID 20871632
Development of an EGFRvIII specific recombinant antibody
EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community.In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAb(DMvIII), specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 10⁷ M⁻¹ as determined by enzyme-linked immunosorbent assay (ELISA).This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.
View details for DOI 10.1186/1472-6750-10-72
View details for Web of Science ID 000283354200001
View details for PubMedID 20925961
View details for PubMedCentralID PMC2959087
MEASURING THE CONSTITUTIVE ACTIVATION OF C-JUN N-TERMINAL KINASE ISOFORMS
METHODS IN ENZYMOLOGY, VOLUME 484: CONSTITUTIVE ACTIVITY IN RECEPTORS AND OTHER PROTEINS, PART A
2010; 484: 531-548
The c-Jun N-terminal kinases (JNK) are important regulators of cell growth, proliferation, and apoptosis. JNKs are typically activated by a sequence of events that include phosphorylation of its T-P-Y motif by an upstream kinase, followed by homodimerization and translocation to the nucleus. Constitutive activation of JNK has been found in a variety of cancers including non-small cell lung carcinomas, gliomas, and mantle cell lymphoma. In vitro studies show that constitutive activation of JNK induces a transformed phenotype in fibroblasts and enhances tumorigenicity in a variety of cell lines. Interestingly, a subset of JNK isoforms was recently found to autoactivate rendering the proteins constitutively active. These constitutively active JNK proteins were found to play a pivotal role in activating transcription factors that increase cellular growth and tumor formation in mice. In this chapter, we describe techniques and methods that have been successfully used to study the three components of JNK activation. Use of these techniques may lead to a better understanding of the components of JNK pathways and how JNK is activated in cancer cells.
View details for DOI 10.1016/S0076-6879(10)84026-1
View details for Web of Science ID 000284508500026
View details for PubMedID 21036249
Constitutive Activity of JNK2 alpha 2 Is Dependent on a Unique Mechanism of MAPK Activation
JOURNAL OF BIOLOGICAL CHEMISTRY
2008; 283 (50): 34935-34945
c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2alpha2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2alpha2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2alpha2 autophosphorylation and autoactivation. We find that JNK2alpha2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the alpha-region. Alanine scanning mutagenesis of the alpha-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2alpha2 dimerization rendering JNK2alpha2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2alpha2 homodimerization. We also show that a kinase-inactive JNK2alpha2 mutant can interact with and inhibit wild type JNK2alpha2 autophosphorylation, suggesting that JNK2alpha2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2alpha2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2alpha2 activity and that prevention of dimerization may decrease JNK2alpha2 induced tumorigenic phenotypes.
View details for DOI 10.1074/jbc.M804970200
View details for Web of Science ID 000261469100050
View details for PubMedID 18940813
View details for PubMedCentralID PMC2596385
Evidence that Proteasome-Dependent Degradation of the Retinoblastoma Protein in Cells Lacking A-Type Lamins Occurs Independently of Gankyrin and MDM2
2007; 2 (9)
A-type lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB) stabilization through unknown mechanism(s). Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna-/- fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna-/- fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna+/+ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16(ink4a)-mediated cell cycle arrest. These findings suggest that lamin A-mediated degradation of pRB would be gankyrin-dependent. However, effective RNAi-enforced reduction of gankyrin expression in Lmna-/- cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2-pRB interaction by transfecting Lmna-/- cells with p14(arf). p14(arf) expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna-/- cells.Our findings suggest that pRB degradation in Lmna-/- cells occurs by gankyrin and MDM2-independent mechanisms, leading us to propose the existence of a third proteasome-dependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna-/- fibroblasts. Second, Lmna-/- cells are refractory to p14(arf)-mediated cell cycle arrest, as was previously shown with p16(ink4a). Potential roles of lamin A/C in the suppression of tumorigenesis are discussed.
View details for DOI 10.1371/journal.pone.0000963
View details for Web of Science ID 000207455800041
View details for PubMedID 17896003
Stabilization of the retinoblastoma protein by A-type nuclear lamins is required for INK4A-mediated cell cycle arrest
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (14): 5360-5372
Mutations in the LMNA gene, which encodes all A-type lamins, including lamin A and lamin C, cause a variety of tissue-specific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that A-type lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16(ink4a), we tested the responsiveness of multiple lamin A/C-depleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of A-type lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16(ink4a)-mediated G(1) arrest. Reintroduction of lamin A, lamin C, or pRB restores p16(ink4a)-responsiveness to Lmna(-/-) cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna(-/-) cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other disease-associated mutants, failed to restore p16(ink4a) responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression.
View details for DOI 10.1128/MCB.02464-05
View details for Web of Science ID 000238918000014
View details for PubMedID 16809772
A-type lamins regulate retinoblastoma protein function by promoting subnuclear localization and preventing proteasomal degradation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (26): 9677-9682
The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G(1) phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that A-type lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna(-/-) cells resemble Rb(-/-) cells, exhibiting altered cell-cycle properties and reduced capacity to undergo cell-cycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cell-cycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation.
View details for DOI 10.1073/pnas.0403250101
View details for Web of Science ID 000222405600030
View details for PubMedID 15210943
View details for PubMedCentralID PMC470734