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Browsing by Author "Hao, Chunhai"
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Item Fibrinogen in the glioblastoma microenvironment contributes to the invasiveness of brain tumor‐initiating cells(Wiley, 2021-09) Dzikowski, Lauren; Mirzaei, Reza; Sarkar, Susobhan; Kumar, Mehul; Bose, Pinaki; Bellail, Anita; Hao, Chunhai; Yong, V. Wee; Pathology and Laboratory Medicine, School of MedicineGlioblastomas (GBMs) are highly aggressive, recurrent, and lethal brain tumors that are maintained via brain tumor-initiating cells (BTICs). The aggressiveness of BTICs may be dependent on the extracellular matrix (ECM) molecules that are highly enriched within the GBM microenvironment. Here, we investigated the expression of ECM molecules in GBM patients by mining the transcriptomic databases and also staining human GBM specimens. RNA levels for fibronectin, brevican, versican, heparan sulfate proteoglycan 2 (HSPG2), and several laminins were high in GBMs compared to normal brain, and this was corroborated by immunohistochemistry. While fibrinogen transcript was at normal level in GBM, its protein immunoreactivity was prominent within GBM tissues. These ECM molecules in tumor specimens were in proximity to, and surrounding BTICs. In culture, fibronectin and pan-laminin induced the adhesion of BTICs onto the plastic substratum. However, fibrinogen increased the size of the BTIC spheres by facilitating the adhesive property, motility, and invasiveness of BTICs. These features of elevated invasiveness were corroborated in resected GBM specimens by the close proximity of fibrinogen with matrix metalloproteinase (MMP)-2 and-9, which are proteases implicated in metastasis. Moreover, the effect of fibrinogen-induced invasiveness was attenuated in BTICs where MMP-2 and -9 have been inhibited with siRNAs or pharmacological inhibitors. Our results implicate fibrinogen in GBM as a mediator of the invasive properties of BTICs, and as a target for therapy to reduce BTIC tumorigenecity.Item HB007 Administration Inhibits LN-229 and Patient-Derived Neurospheroid Glioblastoma Cell Growth With the Degradation of SUMO1 and Cell Cycle Regulator CDK4(2023-07-27) Dougherty, Carson; Wohlford, Reagan; Jung, Sunghan; Hao, ChunhaiBackground and Hypothesis: Glioblastoma is the most common and malignant brain cancer and there is no effective therapy currently available to patients with this malignancy. Small ubiquitin-related modifier 1 (SUMO1) is a key regulator of cancer cell proliferation through its role in its modification of cellular proteins in various human cancers, especially glioblastoma. Degradation of SUMO1 through small molecule degrader, HB007, has been shown to inhibit growth in cancer cell lines and xenografts. Here, we hypothesize that HB007 can inhibit the glioblastoma cell growth through degradation of SUMO1 protein in glioblastoma cells and the cancer stem cell enriched neurospheres. Experimental Design: LN-229 glioblastoma cell viability was measured in response to increasing concentrations of HB007. LN-229 and patient-derived neurospheroid glioblastoma cells were cultured and seeded in 4 different plates at 1000 cells/ml concentrations before being treated with HB007 at increasing concentrations encircling the previously described IC50. Cells were then subjected to a SUMO lysis buffer and analyzed via western blot with antibodies specific to SUMO1, CDK4, and actin. Results: HB007 treated LN-229 cells exhibited an IC50 of 1.470µM. Western blot analysis confirmed the dose dependent reduction in SUMO-1-ylated proteins in HB007 treated cells. A reduction in CDK4 confirmed that cell progression is halted in a dose dependent manner in LN-229 and patient-derived neurospheroid glioblastoma cells when treated with HB007. Specificity of HB007 is towards SUMO1 with no nonspecific degradation of SUMO2/3. Conclusion: The cell growth of LN-229 and patient-derived neurospheroid glioblastoma cells was confirmed, through western blot, to be inhibited in a dose dependent manner by HB007. These results further establish the therapeutic potential of SUMO1 degraders as a novel anticancer drug for glioblastoma therapy. In the future, it is hoped that the bioavailability, potency, and blood brain barrier permeability can be improved to make this drug a potential treatment for patients. Presentation recording available online: https://purl.dlib.indiana.edu/iudl/media/k71n501c5gItem LSD1 and aberrant DNA methylation mediate persistence of enteroendocrine progenitors that support BRAF mutant colorectal cancer(American Association for Cancer Research, 2021) Miller, Samuel A.; Policastro, Robert A.; Sriramkumar, Shruthi; Lai, Tim; Huntington, Thomas D.; Ladaika, Christopher A.; Kim, Daeho; Hao, Chunhai; Zentner, Gabriel E.; O’Hagan, Heather M.; Health Sciences, School of Health and Human SciencesDespite the connection of secretory cells, including goblet and enteroendocrine (EEC) cells, to distinct mucus-containing colorectal cancer histologic subtypes, their role in colorectal cancer progression has been underexplored. Here, our analysis of The Cancer Genome Atlas (TCGA) and single-cell RNA-sequencing data demonstrates that EEC progenitor cells are enriched in BRAF-mutant colorectal cancer patient tumors, cell lines, and patient-derived organoids. In BRAF-mutant colorectal cancer, EEC progenitors were blocked from differentiating further by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of intermediate EECs. Mechanistically, secretory cells and the factors they secrete, such as trefoil factor 3, promoted colony formation and activation of cell survival pathways in the entire cell population. Lysine-specific demethylase 1 (LSD1) was identified as a critical regulator of secretory cell specification in vitro and in a colon orthotopic xenograft model, where LSD1 loss blocks formation of EEC progenitors and reduces tumor growth and metastasis. These findings reveal an important role for EEC progenitors in supporting colorectal cancer. SIGNIFICANCE: This study establishes enteroendocrine progenitors as a targetable population that promotes BRAF-mutant colorectal cancer and can be blocked by LSD1 inhibition to suppress tumor growth.Item PD-1 independent of PD-L1 ligation promotes glioblastoma growth through the NFκB pathway(American Association for the Advancement of Science, 2021-11-05) Mirzaei, Reza; Gordon, Ashley; Zemp, Franz J.; Kumar, Mehul; Sarkar, Susobhan; Luchman, H. Artee; Bellail, Anita C.; Hao, Chunhai; Mahoney, Douglas J.; Dunn, Jeff F.; Bose, Pinaki; Yong, V. Wee; Pathology and Laboratory Medicine, School of MedicineBrain tumor–initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2–containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1– or PD-L1–blocking therapies.Item SUMO1 degrader induces ER stress and ROS accumulation through deSUMOylation of TCF4 and inhibition of its transcription of StarD7 in colon cancer(Wiley, 2023) Zhao, Yin Quan; Jin, Hong Ri; Kim, Daeho; Jung, Sung Han; Liu, Sheng; Wan, Jun; Lo, Ho-Yin; Fu, Xue Qi; Wang, Quan; Hao, Chunhai; Bellail, Anita C.; Pathology and Laboratory Medicine, School of MedicineSmall molecule degraders of small ubiquitin-related modifier 1 (SUMO1) induce SUMO1 degradation in colon cancer cells and inhibits the cancer cell growth; however, it is unclear how SUMO1 degradation leads to the anticancer activity of the degraders. Genome-wide CRISPR-Cas9 knockout screens has identified StAR-related lipid transfer domain containing 7 (StarD7) as a critical gene for the degrader’s anticancer activity. Here, we show that both StarD7 mRNA and protein are overexpressed in human colon cancer and its knockout significantly reduces colon cancer cell growth and xenograft progression. The treatment with the SUMO1 degrader lead compound HB007 reduces StarD7 mRNA and protein levels and increases endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production in colon cancer cells and three-dimensional (3D) organoids. The study further provides a novel mechanism of the compound anticancer activity that SUMO1 degrader-induced decrease of StarD7 occur through degradation of SUMO1, deSUMOylation and degradation of T cell-specific transcription 4 (TCF4) and thereby inhibition of its transcription of StarD7 in colon cancer cells, 3D organoids and patient-derived xenografts (PDX).Item Ubiquitination and degradation of SUMO1 by small-molecule degraders extends survival of mice with patient-derived tumors(AAAS, 2021) Bellail, Anita C.; Jin, Hong Ri; Lo, Ho-Yin; Jung, Sung Han; Hamdouchi, Chafiq; Kim, Daeho; Higgins, Ryan K.; Blanck, Maximilian; le Sage, Carlos; Cross, Benedict C. S.; Li, Jing; Mosley, Amber L.; Wijeratne, Aruna B.; Jiang, Wen; Ghosh, Manali; Zhao, Yin Quan; Hauck, Paula M.; Shekhar, Anantha; Hao, Chunhai; Pathology and Laboratory Medicine, School of MedicineDiscovery of small-molecule degraders that activate ubiquitin ligase–mediated ubiquitination and degradation of targeted oncoproteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cancer cell–based drug screen of the NCI drug-like compounds library that enabled identification of small-molecule degraders of the small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies of analogs of the hit compound CPD1 led to identification of a lead compound HB007 with improved properties and anticancer potency in vitro and in vivo. A genome-scale CRISPR-Cas9 knockout screen identified the substrate receptor F-box protein 42 (FBXO42) of cullin 1 (CUL1) E3 ubiquitin ligase as required for HB007 activity. Using HB007 pull-down proteomics assays, we pinpointed HB007’s binding protein as the cytoplasmic activation/proliferation-associated protein 1 (CAPRIN1). Biolayer interferometry and compound competitive immunoblot assays confirmed the selectivity of HB007’s binding to CAPRIN1. When bound to CAPRIN1, HB007 induced the interaction of CAPRIN1 with FBXO42. FBXO42 then recruited SUMO1 to the CAPRIN1-CUL1-FBXO42 ubiquitin ligase complex, where SUMO1 was ubiquitinated in several of human cancer cells. HB007 selectively degraded SUMO1 in patient tumor–derived xenografts implanted into mice. Systemic administration of HB007 inhibited the progression of patient-derived brain, breast, colon, and lung cancers in mice and increased survival of the animals. This cancer cell–based screening approach enabled discovery of a small-molecule degrader of SUMO1 and may be useful for identifying other small-molecule degraders of oncoproteins.