TFE3-SLC36A1 axis promotes resistance to glucose starvation in kidney cancer cells.

Higher demand for nutrients including glucose is characteristic of cancer. "Starving cancer" has been pursued to curb tumor progression. An intriguing regime is to inhibit glucose transporter GLUT1 in cancer cells. In addition, during cancer progression, cancer cells may suffer from insufficient glucose supply. Yet, cancer cells can somehow tolerate glucose starvation. Uncovering the underlying mechanisms shall shed insight into cancer progression and benefit cancer therapy. TFE3 is a transcription factor known to activate autophagic genes. Physiological TFE3 activity is regulated by phosphorylation-triggered translocation responsive to nutrient status. We recently reported TFE3 constitutively localizes to the cell nucleus and promotes cell proliferation in kidney cancer even under nutrient replete condition. It remains unclear whether and how TFE3 responds to glucose starvation. In this study, we show TFE3 promotes kidney cancer cell resistance to glucose starvation by exposing cells to physiologically relevant glucose concentration. We find glucose starvation triggers TFE3 protein stabilization through increasing its O-GlcNAcylation. Furthermore, through an unbiased functional genomic study, we identify SLC36A1, a lysosomal amino acid transporter, as a TFE3 target gene sensitive to TFE3 protein level. We find SLC36A1 is overexpressed in kidney cancer, which promotes mTOR activity and kidney cancer cell proliferation. Importantly, SLC36A1 level is induced by glucose starvation through TFE3, which enhances cellular resistance to glucose starvation. Suppressing TFE3 or SLC36A1 significantly increases cellular sensitivity to GLUT1 inhibitor in kidney cancer cells. Collectively, we uncover a functional TFE3-SLC36A1 axis that responds to glucose starvation and enhances starvation tolerance in kidney cancer.

YTHDF2 protein stabilization by the deubiquitinase OTUB1 promotes prostate cancer cell proliferation via PRSS8 mRNA degradation.

Prostate cancer is a leading cause of cancer-related mortality in males. Dysregulation of RNA adenine N-6 methylation (m6A) contributes to cancer malignancy. m6A on mRNA may affect mRNA splicing, turnover, transportation, and translation. m6A exerts these effects, at least partly, through dedicated m6A reader proteins, including YTH domain-containing family protein 2 (YTHDF2). YTHDF2 is necessary for development while its dysregulation is seen in various cancers, including prostate cancer. However, the mechanism underlying the dysregulation and function of YTHDF2 in cancer remains elusive. Here, we find that the deubiquitinase OUT domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) increases YTHDF2 protein stability by inhibiting its ubiquitination. With in vivo and in vitro ubiquitination assays, OTUB1 is shown to block ubiquitin transfer to YTHDF2 independent of its deubiquitinase activity. Furthermore, analysis of functional transcriptomic data and m6A-sequencing data identifies PRSS8 as a potential tumor suppressor gene. OTUB1 and YTHDF2 decrease mRNA and protein levels of PRSS8, which is a trypsin-like serine protease. Mechanistically, YTHDF2 binds PRSS8 mRNA and promotes its degradation in an m6A-dependent manner. Further functional study on cellular and mouse models reveals PRSS8 is a critical downstream effector of the OTUB1-YTHDF2 axis in prostate cancer. We find in prostate cancer cells, PRSS8 decreases nuclear ß-catenin level through E-cadherin, which is independent of its protease activity. Collectively, our study uncovers a key regulator of YTHDF2 protein stability and establishes a functional OTUB1-YTHDF2-PRSS8 axis in prostate cancer.

The P53-P21-RB1 pathway promotes BRD4 degradation in liver cancer through USP1.

Liver cancer is notoriously refractory to conventional therapeutics. Tumor progression is governed by the interplay between tumor-promoting genes and tumor-suppressor genes. BRD4, an acetyl lysine-binding protein, is overexpressed in many cancer types, which promotes activation of a pro-tumor gene network. But the underlying mechanism for BRD4 overexpression remains incompletely understood. In addition, understanding the regulatory mechanism of BRD4 protein level will shed insight into BRD4-targeting therapeutics. In this study, we investigated the potential relation between BRD4 protein level and P53, the most frequently dysregulated tumor suppressor. By analyzing the TCGA datasets, we first identify a strong negative correlation between protein levels of P53 and BRD4 in liver cancer. Further investigation shows that P53 promotes BRD4 protein degradation. Mechanistically, P53 indirectly represses the transcription of USP1, a deubiquitinase, through the P21-RB1 axis. USP1 itself is also overexpressed in liver cancer and we show USP1 deubiquitinates BRD4 in vivo and in vitro, which increases BRD4 stability. With cell proliferation assays and xenograft model, we show the pro-tumor role of USP1 is partially mediated by BRD4. With functional transcriptomic analysis, we find the USP1-BRD4 axis upholds expression of a group of cancer-related genes. In summary, we identify a functional P53-P21-RB1-USP1-BRD4 axis in liver cancer.

Monitoring Autophagy with GFP-LC3 Reporter.

Autophagy is a key process that maintains cellular homeostasis. Autophagy contributes to various physiological and pathophysiological processes. Development of methodologies for autophagy detection has greatly facilitated the research on autophagy. Among these methodologies, GFP-LC3 reporter has been popularly used in the literature. In this chapter, we will detail step-by-step the GFP-LC3 reporter protocol we have adapted in our lab. This protocol begins with the generation of lentivirus expressing GFP-LC3. Then, the cells are transduced with titrated virus. After selecting the positive cells, single colonies are isolated, characterized, validated, and used in further study.

TRIM28 represses renal cell carcinoma cell proliferation by inhibiting TFE3/KDM6A-regulated autophagy.

Autophagy plays a pivotal role in physiology and pathophysiology, including cancer. Mechanisms of autophagy dysregulation in cancer remain elusive. Loss of function of TRIM28, a multifunction protein, is seen in familial kidney malignancy, but the mechanism by which TRIM28 contributes to the etiology of kidney malignancy is unclear. In this study, we show TRIM28 retards kidney cancer cell proliferation through inhibiting autophagy. Mechanistically, we find TRIM28 promotes ubiquitination and proteasome-mediated degradation of transcription factor TFE3, which is critical for autophagic gene expression. Genetic activation of TFE3 due to gene fusion is known to cause human kidney malignancy, but whether and how transcription activation by TFE3 involves chromatin changes is unclear. Here, we find another mode of TFE3 activation in human renal carcinoma. We find that TFE3 is constitutively localized to the cell nucleus in human and mouse kidney cancer, where it increases autophagic gene expression and promotes cell autophagy as well as proliferation. We further uncover that TFE3 interacts with and recruits histone H3K27 demethylase KDM6A for autophagic gene upregulation. We reveal that KDM6A contributes to expression of TFE3 target genes through increasing H3K4me3 rather than demethylating H3K27. Collectively, in this study, we identify a functional TRIM28-TFE3-KDM6A signal axis, which plays a critical role in kidney cancer cell autophagy and proliferation.

Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector.

Type VI CRISPR effector Cas13d from Ruminococcus flavefaciens XPD3002 (RfxCas13d) is an RNA-guided RNA endonuclease. RfxCas13d has been harnessed to knockdown gene expression with high specificity in various systems including mammalian cells. While inducible knockdown is advantageous over constitutive knockdown in many scenarios, current inducible systems of RfxCas13d express CRISPR RNA and Cas13d separately. Such systems could be cumbersome to handle and may hamper the application of RfxCas13d in some scenarios. Here, we design an all-in-one Cas13d lentivirus vector which renders efficient and inducible knockdown in a doxycycline dosage-dependent manner. Furthermore, we find that Cas13d has a short half-life in mammalian cells. As a result, knockdown can be promptly reversed after doxycycline withdrawal. This vector is particularly useful for applications involving indispensable genes and/or in cells hard to transduce.

Lysine demethylase KDM1A promotes cell growth via FKBP8-BCL2 axis in hepatocellular carcinoma.

Advanced hepatocellular carcinoma (HCC) has a dismal prognosis. KDM1A (lysine demethylase 1A), overexpressed in multiple cancer types, is a lysine demethylase that targets both histone and nonhistone proteins. However, it is unclear how KDM1A expression affects HCC etiology. Here, we show that KDM1A can interact with and demethylate FKBP8 (FKBP prolyl isomerase 8), a cytoplasmic protein that regulates cell survival through the antiapoptotic protein BCL2 (B-cell lymphoma-2). We show that demethylation of FKBP8 enhances its ability to stabilize BCL2. Consistently, we observed positive correlation between KDM1A and BCL2 protein levels in liver cancer patients. Functionally, we reveal that FKBP8 demethylation by KDM1A is critical for liver cancer cell growth in vitro and in vivo. We went on to explore the mechanisms that might regulate KDM1A cytoplasmic localization. We found that the cytoplasmic localization and protein stability of KDM1A were promoted by acetylation at lysine-117 by the acetyl transferase KAT8 (lysine acetyltransferase 8). In agreement with this, we show that KDM1A-K117 (lysine 117) acetylation promotes demethylation of FKBP8 and level of BCL2. Finally, it has been shown that the efficacy of sorafenib, a first-line treatment for advanced HCC, is limited by clinical resistance. We show that KDM1A and BCL2 protein levels are increased during acquired sorafenib resistance, whereas inhibiting KDM1A can antagonize sorafenib resistance. Collectively, these results define a functional KDM1A-FKBP8-BCL2 axis in HCC.

Versatile functions of RNA m6A machinery on chromatin.

m6A, a conserved and abundant modification on RNA, regulates RNA processing and function. RNA m6A machinery, including writers, erasers, and readers of m6A, is indispensable for m6A installation and function. Intriguingly, recent studies have revealed that m6A machinery can be recruited to chromatin by pleiotropic factors, including nascent RNA, transcription factors, regulatory RNA, histone modifications, and epigenetic machinery. Consequently, recruitment of m6A machinery can directly regulate chromatin biology, such as transcription, DNA damage repair, and DNA recombination beyond installation of m6A on nascent mRNA. Here, we discuss recent evidence showing that m6A machinery is targeted to chromatin and the direct biological consequences along with the underlying mechanisms.

DOT1L O-GlcNAcylation promotes its protein stability and MLL-fusion leukemia cell proliferation.

Histone lysine methylation functions at the interface of the extracellular environment and intracellular gene expression. DOT1L is a versatile histone H3K79 methyltransferase with a prominent role in MLL-fusion leukemia, yet little is known about how DOT1L responds to extracellular stimuli. Here, we report that DOT1L protein stability is regulated by the extracellular glucose level through the hexosamine biosynthetic pathway (HBP). Mechanistically, DOT1L is O-GlcNAcylated at evolutionarily conserved S1511 in its C terminus. We identify UBE3C as a DOT1L E3 ubiquitin ligase promoting DOT1L degradation whose interaction with DOT1L is susceptible to O-GlcNAcylation. Consequently, HBP enhances H3K79 methylation and expression of critical DOT1L target genes such as HOXA9/MEIS1, promoting cell proliferation in MLL-fusion leukemia. Inhibiting HBP or O-GlcNAc transferase (OGT) increases cellular sensitivity to DOT1L inhibitor. Overall, our work uncovers O-GlcNAcylation and UBE3C as critical determinants of DOT1L protein abundance, revealing a mechanism by which glucose metabolism affects malignancy progression through histone methylation.

O-GlcNAcylation links oncogenic signals and cancer epigenetics.

Prevalent dysregulation of epigenetic modifications plays a pivotal role in cancer. Targeting epigenetic abnormality is a new strategy for cancer therapy. Understanding how conventional oncogenic factors cause epigenetic abnormality is of great basic and translational value. O-GlcNAcylation is a protein modification which affects physiology and pathophysiology. In mammals, O-GlcNAcylation is catalyzed by one single enzyme OGT and removed by one single enzyme OGA. O-GlcNAcylation is affected by the availability of the donor, UDP-GlcNAc, generated by the serial enzymatic reactions in the hexoamine biogenesis pathway (HBP). O-GlcNAcylation regulates a wide spectrum of substrates including many proteins involved in epigenetic modification. Like epigenetic modifications, abnormality of O-GlcNAcylation is also common in cancer. Studies have revealed substantial impact on HBP enzymes and OGT/OGA by oncogenic signals. In this review, we will first summarize how oncogenic signals regulate HBP enzymes, OGT and OGA in cancer. We will then integrate this knowledge with the up to date understanding how O-GlcNAcylation regulates epigenetic machinery. With this, we propose a signal axis from oncogenic signals through O-GlcNAcylation dysregulation to epigenetic abnormality in cancer. Further elucidation of this axis will not only advance our understanding of cancer biology but also provide new revenues towards cancer therapy.