Mingfei Yan

A thirty-year quest for a role of R-Ras in cancer: from an oncogene to a multitasking GTPase

Since the identification of R-Ras, which is the first Ras-related GTPase isolated based on sequence similarity to the classical RAS oncogene, more than 160 members of the Ras superfamily of GTPases have been identified and classified into the Ras, Rho, Rap, Rab, Ran, Arf, Rheb, RGK, Rad, Rit, and Miro subfamilies. R-Ras belongs to the Ras subfamily of small G-proteins, which are frequently implicated in cell growth and differentiation. Although the roles of R-Ras in cellular transformation and integrin-mediated cell adhesion have been extensively studied, the physiological function of this enigmatic G-protein was only revealed when a mouse strain deficient in R-Ras was generated. In parallel, a plethora of research findings also linked R-Ras with processes including tumor angiogenesis, axon guidance, and immune cell trafficking. Several upstream factors that modulate R-Ras GTP-binding were identified including Notch, semaphorin, and chemokine C-C motif ligand 21. A review of our evolving understanding of the role of R-Ras in oncogenesis is timely, as this year marks the 30th anniversary of the publication describing the cloning of R-Ras.

R-Ras Regulates Murine T Cell Migration and Intercellular Adhesion Molecule-1 Binding

The trafficking of T-lymphocytes to peripheral draining lymph nodes is crucial for mounting an adaptive immune response. The role of chemokines in the activation of integrins via Ras-related small GTPases has been well established. R-Ras is a member of the Ras-subfamily of small guanosine-5’-triphosphate-binding proteins and its role in T cell trafficking has been investigated in R-Ras null mice (Rras −/−). An examination of the lymphoid organs of Rras −/− mice revealed a 40% reduction in the cellularity of the peripheral lymph nodes. Morphologically, the high endothelial venules of Rras −/− mice were more disorganized and less mature than those of wild-type mice. Furthermore, CD4+ and CD8+ T cells from Rras −/− mice had approximately 42% lower surface expression of L-selectin/CD62L. These aberrant peripheral lymph node phenotypes were associated with proliferative and trafficking defects in Rras −/− T cells. Furthermore, R-Ras could be activated by the chemokine, CCL21. Indeed, Rras −/− T cells had approximately 14.5% attenuation in binding to intercellular adhesion molecule 1 upon CCL21 stimulation. Finally, in a graft-versus host disease model, recipient mice that were transfused with Rras −/− T cells showed a significant reduction in disease severity when compared with mice transplanted with wild-type T cells. These findings implicate a role for R-Ras in T cell trafficking in the high endothelial venules during an effective immune response.

Differential regulation of the pro-inflammatory biomarker, YKL-40/CHI3L1, by PTEN/Phosphoinositide 3-kinase and JAK2/STAT3 pathways in glioblastoma

Constitutive activation of the phosphoinositide 3-kinase/AKT signaling pathway is frequently observed in high-grade gliomas with high frequency of losing PTEN tumor suppressor. To identify transcriptomic profiles associated with a hyperactivated PI3K pathway, RNA-sequencing analysis was performed in a glioblastoma cell line stably expressing PTEN. RNA-sequencing revealed enriched transcripts of pro-inflammatory mediators, and among the genes that displayed high differential expression was the secreted glycoprotein YKL-40. Treatment with chemical inhibitors that target the PI3K/AKT pathway elicited differential effects on YKL-40 expression in selected GBM cell lines, indicating that its expression displayed tumor cell-specific variations. This variability appeared to be correlated with the ability to transactivate the immune signaling molecules JAK2 and STAT3. In summary, the differential expression of the immunomodulatory molecule YKL-40 may affect the treatment efficacy of PI3K/AKT-based pathway inhibitors in glioblastoma.

Loss of tumor suppressor IGFBP4 drives epigenetic reprogramming in hepatic carcinogenesis

Abstract Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.

PTEN PDZ-binding domain suppresses mammary carcinogenesis in the MMTV-PyMT breast cancer model

Phosphatase and tension homolog (PTEN) is a potent tumor suppressor that possesses a PDZ-binding domain (PDZ-BD) at the end of its carboxyl terminus, whose functions during tumorigenesis remains unclear. Here, we crossed a mouse strain with germline deletion of PTEN PDZ-BD with MMTV-PyMT breast cancer model, and found that knockout (KO) mice display normal development of mammary glands, but have both increased breast tumorigenicity and lung metastasis. Orthotopic allograft experiments suggest the loss of PTEN PDZ-BD in breast cancer cells rather than in tumor microenvironment plays a prominent role in increasing tumor burden. Through RNA-sequencing, we observed a significant downregulation of myoepithelial marker genes in both KO primary breast cancer and orthotopic allografts. Moreover, these myoepithelial marker genes are significantly downregulated in human breast cancer tissues, and are associated with poorer clinical prognosis. In addition, several homeobox genes were also identified to be downreguated in KO breast cancer, whose expressions showed significant positive correlation with myoepithelial marker genes. Overall, our findings suggest a novel tumor suppressive role of PTEN PDZ-BD in a murine model of breast cancer, and the mechanism involves the dysregulation of homeobox genes which may result in defective myoepithelial differentiation in breast cancer cells.

Identification of a PTEN mutation with reduced protein stability, phosphatase activity, and nuclear localization in Hong Kong patients with autistic features, neurodevelopmental delays, and macrocephaly

PTEN is a tumor suppressor gene inactivated in over 30% of human cancers. It encodes a lipid phosphatase that serves as a gatekeeper of the phosphoinositide 3‐kinase signaling pathway. Germline mutation frequently occurs in this gene in patients diagnosed with PTEN Hamartoma Tumor Syndrome (PHTS). PHTS individuals are characterized by macrocephaly, benign growth of multiple tissues and increased tumor risk. In addition, autistic phenotypes are found in 10–20% of individuals carrying the germline PTEN mutation with macrocephaly. In this report, 13 suspected PHTS patients were screened for mutation in the PTEN gene. A missense variant (c. 302T > C) substituting the isoleucine at codon 101 to a threonine, a single nucleotide insertion (c. 327‐328insC) causing a frame shift mutation and termination at codon 109, and a nonsense variant (c. 1003C > T) truncated the protein at codon 335 were identified. The I101T mutation significantly reduced PTEN protein expression levels by 2.5‐ to 4.0‐fold. Mechanistically, I101T reduced the protein half‐life of PTEN possibly due to enhanced polyubiquitination at Lysine 13. However, the I101T mutant retained almost 30% of the lipid phosphatase activity of the wild‐type protein. Finally, the I101T mutant has reduced phosphorylation at a PTEN auto‐dephosphorylation site at Threonine 366 and a lowered ratio of nuclear to cytosolic protein level. These partial losses of multiple PTEN biochemical functions may contribute to the tissue overgrowth and autistic features of this PHTS patient. Autism Res 2018, 11: 1098–1109.

Abstract 2163: Mapping SUMOylation sites of PTEN tumor suppressor

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA PTEN tumor suppressor plays crucial function in multiple cellular processes, including proliferation, migration, DNA repair, cell cycle and survival signaling. Its function is tightly controlled by a series of regulatory processes at transcriptional and post-translational levels. Small ubiquitin-related modifier (SUMO) is an essential post-transcriptional modifier which involves in many important biological functions. Previous studies showed that PTEN can be modified by SUMOylation on K254 and K266. SUMOylation on K254 controls the nuclear localization and retention of PTEN and plays essential role in DNA damage repair. On the contrary, SUMOylation on K266 is responsible for its membrane binding, which is required for the down-regulation of PI3K/AKT pathway. However, most studies mainly focus on SUMO1 modification, function of SUMO2/3 involved PTEN regulation is still unclear. In addition, the dynamic regulation and biological activities of different isoforms of SUMOylated PTEN in different subcellular compartments are not well-defined. Also, how PTEN phosphorylation impacted on PTEN SUMOylated have not been addressed fully. To address these questions, a series of PTEN SUMOylation defective-mutants targeted to different subcellular compartments were generated. In addition, a panel of phosphorylation sites mutants (T366A, S370A, S380A, T382A, T383A, and S385A) were included. These mutants were cotransfected with His-tagged SUMO1, SUMO2, and SUMO3 in 293T cells. SUMOylated PTEN protein species are affinity absorbed onto nickel columns. The characterization of these mutants will be discussed. To further delineate the dynamic regulation of SUMOylated PTEN, we are developing a BRET-based live cell imaging system by the ectopic co-expression of a HALO-tagged SUMO and NanoLuc-tagged PTEN expression constructs to monitor the subcellular localization of SUMO-conjugated PTEN in vivo. This work was supported by Hong Kong Research Grant Council (460713) and Hong Kong PhD Fellowship Scheme (PF-12-13876). Citation Format: Yubing Wang, Chiwai Wong, Mingfei Yan, Penelope Or, Andrew M. Chan. Mapping SUMOylation sites of PTEN tumor suppressor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2163. doi:10.1158/1538-7445.AM2015-2163