Browsing by Author "Ratliff, Timothy L."
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Item A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera(Wiley, 2020) Jack, Sherwin; Madhivanan, Kayalvizhi; Ramadesika, Swetha; Subramanian, Sneha; Edwards, Daniel F., II; Elzey, Bennett D.; Dhawan, Deepika; McCluskey, Andrew; Kischuk, Erin M.; Loftis, Alexander R.; Truex, Nicholas; Santos, Michael; Lu, Mike; Rabideau, Amy; Pentelute, Bradley; Collier, John; Kaimakliotis, Hristos; Koch, Michael; Ratliff, Timothy L.; Knapp, Deborah W.; Aguilar, Ruben C.; Urology, School of MedicineBladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.Item Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer(AACR Publications, 2016-09) Vickman, Renee E.; Crist, Scott A.; Kerian, Kevin; Eberlin, Livia; Cooks, R. Graham; Burcham, Grant N.; Buhman, Kimberly K.; Hu, Chang-Deng; Mesecar, Andrew D.; Cheng, Liang; Ratliff, Timothy L.; Pathology and Laboratory Medicine, School of MedicineCholesterol accumulates in prostate lesions and has been linked to prostate cancer (PCa) incidence and progression. However, how accumulated cholesterol contributes to PCa development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared to normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, PCa cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical co-regulators that influence AR activity.Item Cholesterol Sulfotransferase SULT2B1b Modulates Sensitivity to Death Receptor Ligand TNFα in Castration-Resistant Prostate Cancer(American Association for Cancer Research, 2019-06) Vickman, Renee E.; Yang, Jiang; Lanman, Nadia A.; Cresswell, Gregory M.; Zheng, Faye; Zhang, Chi; Doerge, R. W.; Crist, Scott A.; Mesecar, Andrew D.; Hu, Chang-Deng; Ratliff, Timothy L.; Medical and Molecular Genetics, School of MedicineCholesterol sulfotransferase, SULT2B1b, has been demonstrated to modulate both androgen receptor activity and cell growth properties. However, the mechanism(s) by which SULT2B1b alters these properties within prostate cancer cells has not been described. Furthermore, specific advantages of SULT2B1b expression in prostate cancer cells is not understood. In these studies, single-cell mRNA sequencing (scRNA-seq) was conducted to compare the transcriptomes of SULT2B1b knockdown (KD) versus Control KD LNCaP cells. Over 2,000 differentially expressed (DE) genes were identified along with alterations in numerous canonical pathways, including the death receptor signaling pathway. The studies herein demonstrate that SULT2B1b KD increases tumor necrosis factor alpha (TNF) expression in prostate cancer cells and results in NF-κB activation in a TNF-dependent manner. More importantly, SULT2B1b KD significantly enhances TNF-mediated apoptosis in both TNF-sensitive LNCaP cells and TNF-resistant C4–2 cells. Overexpression of SULT2B1b in LNCaP cells also decreases sensitivity to TNF-mediated cell death, suggesting that SULT2B1b modulates pathways dictating the TNF sensitivity capacity of prostate cancer cells. Probing human prostate cancer patient datasets further support this work by providing evidence that SULT2B1b expression is inversely correlated with TNF-related genes, including TNF, CD40LG, FADD, and NFKB1. Together, these data provide evidence that SULT2B1b expression in prostate cancer cells enhances resistance to TNF and may provide a growth advantage. In addition, targeting SULT2B1b may induce an enhanced therapeutic response to TNF treatment in advanced prostate cancer.Item Cholesteryl Ester Accumulation Induced by PTEN Loss and PI3K/AKT Activation Underlies Human Prostate Cancer Aggressiveness(Elsevier, 2014-03-04) Yue, Shuhua; Li, Junjie; Lee, Seung-Young; Lee, Hyeon Jeong; Shao, Tian; Song, Bing; Cheng, Liang; Masterson, Timothy A.; Liu, Xiaoqi; Ratliff, Timothy L.; Cheng, Ji-Xin; Department of Pathology & Laboratory Medicine, IU School of MedicineAltered lipid metabolism is increasingly recognized as a signature of cancer cells. Enabled by label-free Raman spectromicroscopy, we performed quantitative analysis of lipogenesis at single cell level in human patient cancerous tissues. Our imaging data revealed an unexpected, aberrant accumulation of esterified cholesterol in lipid droplets of high-grade prostate cancer and metastases. Biochemical study showed that such cholesteryl ester accumulation was a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway in prostate cancer cells. Furthermore, we found that such accumulation arose from significantly enhanced uptake of exogenous lipoproteins and required cholesterol esterification. Depletion of cholesteryl ester storage significantly reduced cancer proliferation, impaired cancer invasion capability, and suppressed tumor growth in mouse xenograft models with negligible toxicity. These findings open opportunities for diagnosing and treating prostate cancer by targeting the altered cholesterol metabolism.Item Expansion of prostate epithelial progenitor cells after inflammation of the mouse prostate(American Physiological Society, 2015-06-15) Wang, Liang; Zoetemelk, Marloes; Chitteti, Brahmananda R.; Ratliff, Timothy L.; Myers, Jason D.; Srour, Edward F.; Broxmeyer, Hal; Jerde, Travis J.; Department of Pharmacology and Toxicology, IU School of MedicineProstatic inflammation is a nearly ubiquitous pathological feature observed in specimens from benign prostate hyperplasia and prostate cancer patients. The microenvironment of the inflamed prostate is highly reactive, and epithelial hyperplasia is a hallmark feature of inflamed prostates. How inflammation orchestrates epithelial proliferation as part of its repair and recovery action is not well understood. Here, we report that a novel epithelial progenitor cell population is induced to expand during inflammation. We used sphere culture assays, immunofluorescence, and flow cytometry to show that this population is increased in bacterially induced inflamed mouse prostates relative to naïve control prostates. We confirmed from previous reports that this population exclusively possesses the ability to regrow entire prostatic structures from single cell culture using renal grafts. In addition, putative progenitor cells harvested from inflamed animals have greater aggregation capacity than those isolated from naïve control prostates. Expansion of this critical cell population requires IL-1 signaling, as IL-1 receptor 1-null mice exhibit inflammation similar to wild-type inflamed animals but exhibit significantly reduced progenitor cell proliferation and hyperplasia. These data demonstrate that inflammation promotes hyperplasia in the mouse prostatic epithelium by inducing the expansion of a selected epithelial progenitor cell population in an IL-1 receptor-dependent manner. These findings may have significant impact on our understanding of how inflammation promotes proliferative diseases such as benign prostatic hyperplasia and prostate cancer, both of which depend on expansion of cells that exhibit a progenitor-like nature.Item Folate Receptor Beta Designates Immunosuppressive Tumor-Associated Myeloid Cells That Can Be Reprogrammed with Folate-Targeted Drugs(AACR, 2021-02) Cresswell, Gregory M.; Wang, Bingbing; Kischuk, Erin M.; Broman, Meaghan M.; Alfar, Rami A.; Vickman, Renee E.; Dimitrov, Dimiter S.; Kularatne, Sumith A.; Sundaram, Chandru P.; Singhal, Sunil; Eruslanov, Evgeniy B.; Crist, Scott A.; Elzey, Bennett D.; Ratliff, Timothy L.; Low, Philip S.; Urology, School of MedicineAlthough immunotherapies of tumors have demonstrated promise for altering the progression of malignancies, immunotherapies have been limited by an immunosuppressive tumor microenvironment (TME) that prevents infiltrating immune cells from performing their anticancer functions. Prominent among immunosuppressive cells are myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) that inhibit T cells via release of immunosuppressive cytokines and engagement of checkpoint receptors. Here, we explore the properties of MDSCs and TAMs from freshly isolated mouse and human tumors and find that an immunosuppressive subset of these cells can be distinguished from the nonimmunosuppressive population by its upregulation of folate receptor beta (FRβ) within the TME and its restriction to the TME. This FRβ+ subpopulation could be selectively targeted with folate-linked drugs. Delivery of a folate-targeted TLR7 agonist to these cells (i) reduced their immunosuppressive function, (ii) increased CD8+ T-cell infiltration, (iii) enhanced M1/M2 macrophage ratios, (iv) inhibited tumor growth, (v) blocked tumor metastasis, and (vi) improved overall survival without demonstrable toxicity. These data reveal a broadly applicable strategy across tumor types for reprogramming MDSCs and TAMs into antitumorigenic immune cells using a drug that would otherwise be too toxic to administer systemically. The data also establish FRβ as the first marker that distinguishes immunosuppressive from nonimmunosuppressive subsets of MDSCs and TAMs. Because all solid tumors accumulate MDSCs and TAMs, a general strategy to both identify and reprogram these cells should be broadly applied in the characterization and treatment of multiple tumors.Item Identification of LIMK2 as a therapeutic target in castration resistant prostate cancer(Elsevier, 2019-04) Nikhil, Kumar; Chang, Lei; Viccaro, Keith; Jacobsen, Max; McGuire, Callista; Satapathy, Shakti R.; Tandiary, Michael; Broman, Meaghan M.; Cresswell, Gregory; He, Yizhou J.; Sandusky, George E.; Ratliff, Timothy L.; Chowdhury, Dipanjan; Shah, Kavita; Pathology and Laboratory Medicine, School of MedicineThis study identified LIMK2 kinase as a disease-specific target in castration resistant prostate cancer (CRPC) pathogenesis, which is upregulated in response to androgen deprivation therapy, the current standard of treatment for prostate cancer. Surgical castration increases LIMK2 expression in mouse prostates due to increased hypoxia. Similarly, human clinical specimens showed highest LIMK2 levels in CRPC tissues compared to other stages, while minimal LIMK2 was observed in normal prostates. Most notably, inducible knockdown of LIMK2 fully reverses CRPC tumorigenesis in castrated mice, underscoring its potential as a clinical target for CRPC. We also identified TWIST1 as a direct substrate of LIMK2, which uncovered the molecular mechanism of LIMK2-induced malignancy. TWIST1 is strongly associated with CRPC initiation, progression and poor prognosis. LIMK2 increases TWIST1 mRNA levels upon hypoxia; and stabilizes TWIST1 by direct phosphorylation. TWIST1 also stabilizes LIMK2 by inhibiting its ubiquitylation. Phosphorylation-dead TWIST1 acts as dominant negative and fully prevents EMT and tumor formation in vivo, thereby highlighting the significance of LIMK2-TWIST1 signaling axis in CRPC. As LIMK2 null mice are viable, targeting LIMK2 should have minimal collateral toxicity, thereby improving the overall survival of CRPC patients.Item Inflammation impacts androgen receptor signaling in basal prostate stem cells through interleukin 1 receptor antagonist(Springer Nature, 2024-10-25) Cooper, Paula O.; Yang, Jiang; Wang, Hsing-Hui; Broman, Meaghan M.; Jayasundara, Shyaman Madhawa; Sahoo, Subhransu Sekhar; Yan, Bingyu; Awdalkreem, Gada D.; Cresswell, Gregory M.; Wang, Liang; Goossens, Emery; Lanman, Nadia A.; Doerge, Rebecca W.; Zheng, Faye; Cheng, Liang; Alqahtani, Saeed; Crist, Scott A.; Braun, Robert E.; Kazemian, Majid; Jerde, Travis J.; Ratliff, Timothy L.; Microbiology and Immunology, School of MedicineChronic prostate inflammation in patients with benign prostate hyperplasia (BPH) correlates with the severity of symptoms. How inflammation contributes to prostate enlargement and/or BPH symptoms and the underlying mechanisms remain unclear. In this study, we utilize a unique transgenic mouse model that mimics chronic non-bacterial prostatitis in men and investigate the impact of inflammation on androgen receptor (AR) in basal prostate stem cells (bPSC) and their differentiation in vivo. We find that inflammation significantly enhances AR levels and activity in bPSC. More importantly, we identify interleukin 1 receptor antagonist (IL-1RA) as a crucial regulator of AR in bPSC during inflammation. IL-1RA is one of the top molecules upregulated by inflammation, and inhibiting IL-1RA reverses the enhanced AR activity in organoids derived from inflamed bPSC. Additionally, IL-1RA appears to activate AR by counteracting IL-1α's inhibitory effect. Furthermore, using a lineage tracing model, we observe that inflammation induces bPSC proliferation and differentiation into luminal cells even under castrate conditions, indicating that AR activation driven by inflammation is sufficient to promote bPSC proliferation and differentiation. Taken together, our study uncovers mechanisms through which inflammation modulates AR signaling in bPSC and induces bPSC luminal differentiation that may contribute to prostate hyperplasia.Item Inflammation Impacts Androgen Receptor Signaling in Basal Prostate Stem Cells Through Interleukin 1 Receptor Antagonist(Research Square, 2023-12-15) Cooper, Paula O.; Yang, Jiang; Wang, Hsing-Hui; Broman, Meaghan M.; Awdalkreem, Gada D.; Cresswell, Gregory M.; Wang, Liang; Goossens, Emery; Lanman, Nadia A.; Doerge, Rebecca W.; Zheng, Faye; Cheng, Liang; Crist, Scott A.; Braun, Robert E.; Jerde, Travis J.; Ratliff, Timothy L.; Pharmacology and Toxicology, School of MedicineThe majority of patients with benign prostate hyperplasia (BPH) exhibit chronic prostate inflammation and the extent of inflammation correlates with the severity of symptoms. How inflammation contributes to prostate enlargement and/or BPH symptoms and the underlying mechanisms are not clearly understood. We established a unique mouse model Prostate Ovalbumin Expressing Transgenic 3 (POET3) that mimics chronic non-bacterial prostatitis in men to study the role of inflammation in prostate hyperplasia. After the injection of ovalbumin peptide-specific T cells, POET3 prostates exhibited an influx of inflammatory cells and an increase in pro-inflammatory cytokines that led to epithelial and stromal hyperplasia. We have previously demonstrated with the POET3 model that inflammation expands the basal prostate stem cell (bPSC) population and promotes bPSC differentiation in organoid cultures. In this study, we investigated the mechanisms underlying the impact of inflammation on bPSC. We found that AR activity was enhanced in inflamed bPSC and was essential for bPSC differentiation in organoid cultures. Most importantly, we identified, for the first time, interleukin 1 receptor antagonist (IL-1RA) as a key regulator of AR in basal stem cells. IL-1RA was one of the top genes upregulated by inflammation and inhibition of IL-1RA abrogated the enhanced AR nuclear accumulation and activity in organoids derived from inflamed bPSC. The mirroring effects of IL-1RA recombinant protein and IL-1α neutralizing antibody suggest that IL-1RA may function by antagonizing IL-1α inhibition of AR expression. Furthermore, we established a lineage tracing model to follow bPSC during inflammation and under castrate conditions. We found that inflammation induced bPSC proliferation and differentiation into luminal cells even under castrate conditions, indicating that AR activation driven by inflammation in bPSC is sufficient for their proliferation and differentiation under androgen-deprived conditions. However, proliferation of the differentiated bPSC in the luminal layer significantly diminished with castration, suggesting inflammation may not maintain AR activity in stromal cells, as stromal cells deprived of androgen after castration could no longer provide paracrine growth factors essential for luminal proliferation. Taken together, we have discovered novel mechanisms through which inflammation modulates AR signaling in bPSC and induces bPSC luminal differentiation that contributes to prostate hyperplasia.Item Novel Use of Folate-Targeted Intraoperative Fluorescence, OTL38, in Robot-Assisted Laparoscopic Partial Nephrectomy: Report of the First Three Cases(Mary Ann Liebert, 2016) Shum, Cheuk Fan; Bahler, Clinton D.; Low, Philip S.; Ratliff, Timothy L.; Kheyfets, Steven V.; Natarajan, Jay P.; Sandusky, George E.; Sundaram, Chandru P.; Department of Urology, IU School of MedicinePartial nephrectomy is now the preferred surgical option for small renal tumors because it allows nephron preservation without compromising oncologic clearance. Its outcomes depend on the surgeon's ability to continuously identify the edges of the tumor during resection, thus leaving an adequate margin around the tumor without excessive removal of normal parenchyma, as well as keeping a short ischemic time. Folate receptors are highly abundant in the normal kidney, and there is a difference in folate receptor expression between malignant and normal renal tissues. Thus, the use of fluorescent agents that target folate receptors should result in differential fluorescence between the tumor and surrounding parenchyma during partial nephrectomy, which, in turn, helps tumor demarcation for identification and resection. A phase 2 study on the novel use of OTL38 in robot-assisted laparoscopic partial nephrectomy is currently in progress in our institution. The outcomes of the first three cases have shown the possible advantages of OTL38 in intraoperative tumor identification before resection and recognition of residual disease in the surrounding parenchyma after resection. The tumors typically appeared dark while the surrounding parenchyma showed brighter fluorescence. Immediately after tumor resection, the margins of all the specimens appeared to have a uniformly bright fluorescence, suggestive of an intact margin of normal renal parenchyma along the plane of excision. The pattern of intraoperative fluorescence correlates well with immunohistochemistry. No OTL38-related adverse effects have been seen among these three patients. We present the outcomes of these three cases, illustrated with intraoperative and immunohistochemistry images.