H3F3A K27M Mutations Drives a Repressive Transcriptome by Modulating Chromatin Accessibility, Independent of H3K27me3 in Diffuse Midline Glioma.

Background: Heterozygous histone H3.3K27M mutation is a primary oncogenic driver of Diffuse Midline Glioma (DMG). H3.3K27M inhibits the Polycomb Repressive Complex 2 (PRC2) methyltransferase complex, leading to a global reduction and redistributing of the repressive H3 lysine 27 tri-methylation. This rewiring of the epigenome is thought to promote gliomagenesis. Methods: We established novel, isogenic DMG patient-derived cell lines that have been CRISPR-Cas9 edited to H3.3 WT or H3.3K27M alone and in combination with EZH2 and EZH1 co-deletion, inactivating PRC2 methyltransferase activity of PRC2 and eliminating H3K27me3. Results: RNA-seq and ATAC-seq analysis of these cells revealed that K27M has a novel epigenetic effect that appears entirely independent of its effects on PRC2 function. While the loss of the PRC2 complex led to a systemic induction of gene expression (including HOX gene clusters) and upregulation of biological pathways, K27M led to a balanced gene deregulation but having an overall repressive effect on the biological pathways. Importantly, the genes uniquely deregulated by the K27M mutation, independent of methylation loss, are closely associated with changes in chromatin accessibility, with upregulated genes becoming more accessible. Notably, the PRC2- independent function of K27M appears necessary for tumorigenesis as xenografts of our H3.3K27M/EZH1/2 WT cells developed into tumors, while H3.3/EZH1/2 KO cells did not. Conclusion: We demonstrate that K27M mutation alters chromatin accessibility and uniquely deregulates genes, independent of K27 methylation. We further show the mutation's role in altering biological pathways and its necessity for tumor development. Key Points: We revealed genes regulated by H3.3K27M mutation and PRC2 in DMG.H3.3K27M mutation alters chromosome accessibility independent of H3K27me3.PRC2-independent effects of K27M mutation are crucial for tumor development. Importance of the Study: This study is the first to demonstrate that H3F3A K27M mutations drive a repressive transcriptome by modulating chromatin accessibility independently of H3K27 trimethylation in Diffuse Midline Glioma (DMG). By isolating the effects of H3.3 K27me3 loss from those of the K27M mutation, we identified common and unique genes and pathways affected by each. We found that genes uniquely deregulated by K27M showed increased chromatin accessibility and upregulated gene expression, unlike other gene subsets affected by PRC2 knockout. Importantly, we determined the PRC2-independent function of K27M is also essential for tumorigenesis, as xenografts of H3.3 K27M/PRC2 WT cell lines formed tumors, while H3.3WT/PRC2 WT and K27M/PRC2 knockout cells did not. This research builds upon and advances prior studies, such as those identifying EZH2 as a therapeutic target in H3.3K27M DMGs, by revealing critical new pathways for gliomagenesis. The translational significance lies in identifying novel therapeutic targets against this aggressive pediatric cancer.

Using Microinjection of Mammalian Cultured Cells to Study Cell Division.

The introduction of macromolecules directly into individual cells by microinjection is an important technique for manipulating mitotic cells. mRNA, purified proteins, or concentrated antibodies can all be injected directly into a single cell, and their effects monitored by live-cell imaging. The equipment necessary is relatively simple, and the technique can be easily mastered. Here we describe our microinjection setup, how to microinject cultured mammalian cells in mitosis, and how to analyze those cells by same-cell live and fixed imaging.

Manipulating cultured mammalian cells for mitosis research.

The study of mitosis has always relied on bulk-preparation biochemistry techniques (Mazia & Dan, 1952), but very early on lent itself to living, single cell microscopic techniques (Inoue, 1953; Taylor, 1959). Here we describe several of the methods used by our lab to study cell division in living cultured cells, including cold-induced mitotic arrest, cold-induced chromosome missegregation, same-cell live and fixed cell imaging, and microinjection of inactivating antibodies. We detail our imaging system based on an upright fluorescent microscope and spinning disk confocal, as well as the customized "HEKS" metal support slide imaging chambers.

Microsurgery and microinjection techniques in mitosis research.

The use of microtechnique for studying cell division is well established (Begg & Ellis, 1979; Wadsworth, 1999; Zhang & Nicklas, 1999). The advantage of microinjection in cell division research is the timed delivery of a macromolecules at a particular stage of mitosis (for example, pre- vs postanaphase), which can circumvent the spindle assembly checkpoint (Hinchcliffe et al., 2016). Micromanipulation can be used to remove whole organelles, such as the centrosome or nucleus and examine the effects on cell division (Hinchcliffe et al., 2001; Hornick et al., 2011). The focus of this chapter is on methods for microinjection and micromanipulation of cultured mammalian cells. We describe pulling and shaping microneedles, as well as the imaging chambers we use. We also provide information on cell culture conditions, and imaging techniques used for our long-term observation studies, which allow cells to be followed on the order of several days.

LTA4H regulates cell cycle and skin carcinogenesis.

Leukotriene A4 hydrolase (LTA4H), a bifunctional zinc metallo-enzyme, is reportedly overexpressed in several human cancers. Our group has focused on LTA4H as a potential target for cancer prevention and/or therapy. In the present study, we report that LTA4H is a key regulator of cell cycle at the G0/G1 phase acting by negatively regulating p27 expression in skin cancer. We found that LTA4H is overexpressed in human skin cancer tissue. Knocking out LTA4H significantly reduced skin cancer development in the 7,12-dimethylbenz(a)anthracene (DMBA)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted two-stage skin cancer mouse model. LTA4H depletion dramatically decreased anchorage-dependent and -independent skin cancer cell growth by inducing cell cycle arrest at the G0/G1 phase. Moreover, our findings showed that depletion of LTA4H enhanced p27 protein stability, which was associated with decreased phosphorylation of CDK2 at Thr160 and inhibition of the CDK2/cyclin E complex, resulting in down-regulated p27 ubiquitination. These findings indicate that LTA4H is critical for skin carcinogenesis and is an important mediator of cell cycle and the data begin to clarify the mechanisms of LTA4H's role in cancer development.

Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation.

Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed.

Kaempferol targets RSK2 and MSK1 to suppress UV radiation-induced skin cancer.

Solar UV (SUV) irradiation is a major factor in skin carcinogenesis, the most common form of cancer in the United States. The MAPK cascades are activated by SUV irradiation. The 90 kDa ribosomal S6 kinase (RSK) and mitogen and stress-activated protein kinase (MSK) proteins constitute a family of protein kinases that mediate signal transduction downstream of the MAPK cascades. In this study, phosphorylation of RSK and MSK1 was upregulated in human squamous cell carcinoma (SCC) and SUV-treated mouse skin. Kaempferol, a natural flavonol, found in tea, broccoli, grapes, apples, and other plant sources, is known to have anticancer activity, but its mechanisms and direct target(s) in cancer chemoprevention are unclear. Kinase array results revealed that kaempferol inhibited RSK2 and MSK1. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations showed that kaempferol suppresses RSK2 and MSK1 kinase activities to attenuate SUV-induced phosphorylation of cAMP-responsive element binding protein (CREB) and histone H3 in mouse skin cells. Kaempferol was a potent inhibitor of SUV-induced mouse skin carcinogenesis. Further analysis showed that skin from the kaempferol-treated group exhibited a substantial reduction in SUV-induced phosphorylation of CREB, c-Fos, and histone H3. Overall, our results identify kaempferol as a safe and novel chemopreventive agent against SUV-induced skin carcinogenesis that acts by targeting RSK2 and MSK1.

Naproxen induces cell-cycle arrest and apoptosis in human urinary bladder cancer cell lines and chemically induced cancers by targeting PI3K.

Naproxen [(S)-6-methoxy-α-methyl-2-naphthaleneacetic acid] is a potent nonsteroidal anti-inflammatory drug that inhibits both COX-1 and COX-2 and is widely used as an over-the-counter medication. Naproxen exhibits analgesic, antipyretic, and anti-inflammatory activities. Naproxen, as well as other nonsteroidal anti-inflammatory drug, has been reported to be effective in the prevention of urinary bladder cancer in rodents. However, potential targets other than the COX isozymes have not been reported. We examined potential additional targets in urinary bladder cancer cells and in rat bladder cancers. Computer kinase profiling results suggested that phosphoinositide 3-kinase (PI3K) is a potential target for naproxen. In vitro kinase assay data revealed that naproxen interacts with PI3K and inhibits its kinase activity. Pull-down binding assay data confirmed that PI3K directly binds with naproxen in vitro and ex vivo. Western blot data showed that naproxen decreased phosphorylation of Akt, and subsequently decreased Akt signaling in UM-UC-5 and UM-UC-14 urinary bladder cancer cells. Furthermore, naproxen suppressed anchorage-independent cell growth and decreased cell viability by targeting PI3K in both cell lines. Naproxen caused an accumulation of cells at the G1 phase mediated through cyclin-dependent kinase 4, cyclin D1, and p21. Moreover, naproxen induced significant apoptosis, accompanied with increased levels of cleaved caspase-3, caspase-7, and PARP in both cell types. Naproxen-induced cell death was mainly because of apoptosis in which a prominent downregulation of Bcl-2 and upregulation of Bax were involved. Naproxen also caused apoptosis and inhibited Akt phosphorylation in rat urinary bladder cancers induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine.

Autophagy and cellular senescence mediated by Sox2 suppress malignancy of cancer cells.

Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the "stemness" of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, including intracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16(INK4a), p21 and phosphorylated p53 (Ser15). Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers.

Norathyriol suppresses skin cancers induced by solar ultraviolet radiation by targeting ERK kinases.

Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G(2)-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-κB during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

T-LAK cell-originated protein kinase (TOPK) phosphorylation of MKP1 protein prevents solar ultraviolet light-induced inflammation through inhibition of the p38 protein signaling pathway.

Solar UV radiation is a major environmental factor that causes DNA damage, inflammation, and even skin cancer. T-LAK cell-originated protein kinase (TOPK) is expressed widely in both normal and cancer cells and functions to inhibit apoptosis and promote carcinogenesis. However, its function in inflammation is not known. The p38 MAPK signaling pathway plays an important role in solar UV light-induced inflammation. In this study, we found that TOPK negatively regulated the activity of p38α by phosphorylating the p38α-specific phosphatase MKP1 and enhancing the stability of MKP1. Notably, the absence of TOPK in mice resulted in a striking increase in skin inflammation. Therefore, we conclude that TOPK has a protective function in solar UV light-induced inflammation.

Lapatinib, a preventive/therapeutic agent against mammary cancer, suppresses RTK-mediated signaling through multiple signaling pathways.

Activation of receptor tyrosine kinases (RTK) plays a key role in the prognosis of mammary cancer. Lapatinib is a small molecule dual RTK inhibitor that targets epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). Identifying the protein targets involved in the effects of lapatinib and other RTK inhibitors might help determine why preventive efficacy varies. In this study, female Sprague-Dawley rats were given methylnitrosourea (MNU) by intravenous injection resulting in the development of multiple estrogen receptor-positive tumors. Treatment with lapatinib beginning 5 days after MNU was highly effective in preventing cancer development. In addition, we treated rats with palpable mammary tumors with lapatinib daily. In these tumor-bearing animals, treatment continued for 42 days and therapeutic results were obtained. Some rats bearing cancers were treated for 5 days, and the resulting lesions were examined for biomarker modulation. Lapatinib effectively suppressed the abundance of HER2, phosphorylated HER2 (Tyr1221/1222), and phosphorylated EGFR (Tyr1173, Tyr1110) compared with tumors from untreated rats. Protein array analyses allowed parallel determination of the effect of lapatinib on the relative levels of protein phosphorylation and proteins associated with apoptosis. These results combined with immunoreactivity data indicated that, in addition to EGFR and HER2, lapatinib treatment was associated with changes in a number of other signaling molecules, including IGF-1R, Akt, and downstream targets such as GSK3, p27, p53, and cyclin D1 presumably leading to impaired proliferation, apoptosis, or cell-cycle arrest.

TRPV1-antagonist AMG9810 promotes mouse skin tumorigenesis through EGFR/Akt signaling.

In addition to capsaicin, a transient receptor potential channel vanilloid subfamily 1 (TRPV1) agonist, two kinds of antagonists against this receptor are used as therapeutic drugs for pain relief. Indeed, a number of small molecule TRPV1 antagonists are currently undergoing Phase I/II clinical trials to determine their effect on relieving chronic inflammatory pain and migraine headache pain. However, we previously reported that the absence of TRPV1 in mice results in a striking increase in skin carcinogenesis, suggesting that chronic blockade of TRPV1 might increase the risk of tumor development. In this study, we found that a typical TRPV1 antagonist, AMG9810, promotes mouse skin tumor development. The topical application of AMG9810 resulted in a significant increase in the expression level of the epidermal growth factor receptor (EGFR) and its downstream Akt/mammalian target of rapamycin (mTOR)-signaling pathway. This increase was not only observed in AMG9810-treated tumor tissue but was also found in skin tissue treated with AMG9810. In telomerase-immortalized primary human keratinocytes, AMG9810 promoted proliferation that was mediated through the EGFR/Akt/mTOR-signaling pathway. In summary, our data suggest that the TRPV1 antagonist, AMG9810, promotes mouse skin tumorigenesis mediated through EGFR/Akt/mTOR signaling. Thus, the application of this compound for pain relief might increase the risk of skin cancer.