Death effector domain-containing protein induces vulnerability to cell cycle inhibition in triple-negative breast cancer

Lacking targetable molecular drivers, triple-negative breast cancer (TNBC) is the most clinically challenging subtype of breast cancer. In this study, we reveal that Death Effector Domain-containing DNA-binding protein (DEDD), which is overexpressed in > 60% of TNBCs, drives a mitogen-independent G1/S cell cycle transition through cytoplasm localization. The gain of cytosolic DEDD enhances cyclin D1 expression by interacting with heat shock 71 kDa protein 8 (HSC70). Concurrently, DEDD interacts with Rb family proteins and promotes their proteasome-mediated degradation. DEDD overexpression renders TNBCs vulnerable to cell cycle inhibition. Patients with TNBC have been excluded from CDK 4/6 inhibitor clinical trials due to the perceived high frequency of Rb-loss in TNBCs. Interestingly, our study demonstrated that, irrespective of Rb status, TNBCs with DEDD overexpression exhibit a DEDD-dependent vulnerability to combinatorial treatment with CDK4/6 inhibitor and EGFR inhibitor in vitro and in vivo. Thus, our study provided a rationale for the clinical application of CDK4/6 inhibitor combinatorial regimens for patients with TNBC

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111127/1/pmic8033.pd

GAD1 Upregulation Programs Aggressive Features of Cancer Cell Metabolism in the Brain Metastatic Microenvironment

The impact of altered amino acid metabolism on cancer progression is not fully understood. We hypothesized that a metabolic transcriptome shift during metastatic evolution is crucial for brain metastasis. Here, we report a powerful impact in this setting caused by epigenetic upregulation of glutamate decarboxylase 1 (GAD1), a regulator of the GABA neurotransmitter metabolic pathway. In cell-based culture and brain metastasis models, we found that downregulation of the DNA methyltransferase DNMT1 induced by the brain microenvironment-derived clusterin resulted in decreased GAD1 promoter methylation and subsequent upregulation of GAD1 expression in brain metastatic tumor cells. In a system to dynamically visualize cellular metabolic responses mediated by GAD1, we monitored the cytosolic NADH:NAD+ equilibrium in tumor cells. Reducing GAD1 in metastatic cells by primary glia cell coculture abolished the capacity of metastatic cells to utilize extracellular glutamine, leading to cytosolic accumulation of NADH and increased oxidative status. Similarly, genetic or pharmacologic disruption of the GABA metabolic pathway decreased the incidence of brain metastasis in vivo Taken together, our results show how epigenetic changes in GAD1 expression alter local glutamate metabolism in the brain metastatic microenvironment, contributing to a metabolic adaption that facilitates metastasis outgrowth in that setting

Integrative Bioinformatic and Experimental Investigation of the Mechanisms of Brain Metastatic Adaptation

Cancer metastasis alters many of the processes that are necessary for tumorigenesis to facilitate successful metastases. However, a global analysis of the transcriptome landscape of the metastatic tumor has not been well studied. In my analysis, I compared the primary tumor to the brain metastatic tumor to identify the occurring functional transcriptome changes. I further identified the changes that are unique to the brain metastatic process. I finally analyzed the gene expression pattern that occurs between the colonization stage and outgrowth metastatic stage. This analysis provides a global view of the brain metastatic process starting from the primary tumor to early colonization stage and finally to the overt late stage brain metastatic tumor. In conjunction with the analysis of the metastatic tumor, I also conducted a global analysis of the surrounding microenvironment. I first compared the primary tumor and brain metastatic microenvironments for their respective gene expression changes. Furthermore, I compared the surrounding microenvironment during the early and late stages of brain metastases. This global analysis provides one of the first global views of the transcriptome landscape of the brain microenvironment during the entire metastatic process. My global analysis of both the tumor and stroma provide insights into many aspects of the metastatic process. To further investigate these insights, I focused on the role of metabolism in the brain metastatic process. Based on a distinct metabolic transcriptome profile in brain metastases compared to their primary tumor counterparts, I hypothesized that metabolic transcriptome shifting during metastatic evolution is crucial for metastatic success to the brain. Here I show that the expression of the GABA metabolic pathway mediator glutamate decarboxylase 1 (GAD1) is significantly up]regulated. Down]regulation of DNA methyltransferase 1 (DNMT1) induced by the brain microenvironment results in decreased GAD1 promoter methylation and subsequent up–regulation of GAD1 expression. I utilized the Peredox biosensor coupled with time–lapse imaging to monitor the cytosolic NADH:NAD+ equilibrium in tumor cells. Knocking down of GAD1 abolishes the capability of tumor cells to utilize extracellular glutamine. Repurposing of a neurological drug, vigabatrin, results in a significant decrease in brain metastasis incidence. Taken together, my results demonstrated that brain microenvironment–specific metabolic shifting through GAD1 promoter demethylation contributes to the context–dependent cellular metabolic adaption and facilitate brain metastasis outgrowth

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111127/1/pmic8033.pd

Transcription factor network analysis based on single cell RNA-seq identifies that Trichostatin-a reverses docetaxel resistance in prostate Cancer

Abstract Background Overcoming drug resistance is critical for increasing the survival rate of prostate cancer (PCa). Docetaxel is the first cytotoxic chemotherapeutical approved for treatment of PCa. However, 99% of PCa patients will develop resistance to docetaxel within 3 years. Understanding how resistance arises is important to increasing PCa survival. Methods In this study, we modeled docetaxel resistance using two PCa cell lines: DU145 and PC3. Using the Passing Attributes between Networks for Data Assimilation (PANDA) method to model transcription factor (TF) activity networks in both sensitive and resistant variants of the two cell lines. We identified edges and nodes shared by both PCa cell lines that composed a shared TF network that modeled changes which occur during acquisition of docetaxel resistance in PCa. We subjected the shared TF network to connectivity map analysis (CMAP) to identify potential drugs that could disrupt the resistant networks. We validated the candidate drug in combination with docetaxel to treat docetaxel-resistant PCa in both in vitro and in vivo models. Results In the final shared TF network, 10 TF nodes were identified as the main nodes for the development of docetaxel resistance. CMAP analysis of the shared TF network identified trichostatin A (TSA) as a candidate adjuvant to reverse docetaxel resistance. In cell lines, the addition of TSA to docetaxel enhanced cytotoxicity of docetaxel resistant PCa cells with an associated reduction of the IC50 of docetaxel on the resistant cells. In the PCa mouse model, combination of TSA and docetaxel reduced tumor growth and final weight greater than either drug alone or vehicle. Conclusions We identified a shared TF activity network that drives docetaxel resistance in PCa. We also demonstrated a novel combination therapy to overcome this resistance. This study highlights the usage of novel application of single cell RNA-sequencing and subsequent network analyses that can reveal novel insights which have the potential to improve clinical outcomes.http://deepblue.lib.umich.edu/bitstream/2027.42/173543/1/12885_2021_Article_9048.pd

Bone Marrow Macrophages Induce Inflammation by Efferocytosis of Apoptotic Prostate Cancer Cells via HIF-1α Stabilization

The clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow (BM) macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment in their cellular response to hypoxia. Here, we show that BM macrophage efferocytosis increased hypoxia inducible factor-1alpha (HIF-1α) and STAT3 phosphorylation (p-STAT3 at Tyr705) under normoxic conditions, while inhibitors of p-STAT3 reduced HIF-1α. Efferocytosis promoted HIF-1α stabilization, reduced its ubiquitination, and induced HIF-1α and p-STAT3 nuclear translocation. HIF-1α stabilization in efferocytic BM macrophages resulted in enhanced expression of pro-inflammatory cytokine MIF, whereas BM macrophages with inactive HIF-1α reduced MIF expression upon efferocytosis. Stabilization of HIF-1α using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in BM macrophages. Furthermore, BM macrophages treated with recombinant MIF protein activated NF-κB (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by BM macrophages triggers p-STAT3/HIF-1α/MIF signaling to promote further inflammation in the bone tumor microenvironment where a significant number of apoptotic cancer cells are present

Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth

Mechanisms governing adaptation of breast cancer to the brain metastatic microenvironment are unclear. Here, the authors use RNA-sequencing and Drosophila screening to identify Rab11b-mediated endosomal recycling as a unique mechanism for adaptation to a challenging metastatic microenvironment, which can be exploited by repurposing statins