Identification of LIMK2 as a therapeutic target in castration resistant prostate cancer

This 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

Folate Receptor Beta Designates Immunosuppressive Tumor-Associated Myeloid Cells That Can Be Reprogrammed with Folate-Targeted Drugs

Although 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

Inflammation and Benign Prostatic Hyperplasia: Role of Immune Cells and their Interactions in Chronic Inflammation and Cellular Hyperplasia

Benign prostatic hyperplasia (BPH) is a common urologic condition among older men, affecting approximately half of men by age 50 and nearly 80% by age 80. Lower urinary tract symptoms (LUTS) associated with BPH may significantly impact quality of life for many of these men. Inflammation has been associated with the development and progression of BPH however, the precise impact and role(s) of immune cells in these conditions remains unclear. Many previous studies over the decades have explored the roles of immune cells in prostate disease in animal models and prostate tissues from human patients, and, more recently, through transcriptomic analyses of bulk cell populations and of single cells. These and other emerging technologies continue to add to the body of knowledge related to this area. The prostate is a complex organ composed of multiple epithelial and mesenchymal cell types and subtypes. The growth, morphology, and function of these cells is influenced by autocrine and paracrine cell-cell interactions in ways that are largely not yet understood. A better understanding of the composition, heterogeneity, morphology, interactions, and functional features of various prostate cell types, particularly involving immune cells in the context of inflammatory processes, is expected to improve our understanding of the impact of altered cellular composition and communication on prostate homeostasis and disease. Inflammation has been shown to impact the growth, morphology, and function of various prostate cell types. It is hypothesized that inflammation promotes epithelial cell proliferation and differentiation in BPH despite androgen-targeted therapy. It is hypothesized that communications between and within various immune cell populations perpetuate the non-resolving inflammatory microenvironment that promotes prostate cell expansion. In this research, the POET-3 mouse model of inducible autoimmune inflammation is used to evaluate the impact of autoimmune-type inflammation on basal epithelial cell progenitor growth and differentiation in the absence of androgens mimicking the conditions of androgen deprivation therapy (ADT), and to demonstrate the enhanced growth and differentiation potential conferred on basal progenitors by inflammation. Additionally, this research evaluates the morphology, gene expression, and cell-cell interaction predictions of BPH prostate immune cells to explore the role of immune cells and their interactions in driving BPH inflammation. Overall, inflammation induced epithelial and stromal expansion and basal progenitor cell proliferation in vivo and promoted basal progenitor cell growth and differentiation in vitro under androgen-deficient conditions mimicking androgen-targeted therapy. Histologic evaluation of BPH specimens reveals the composition and distribution of immune cells, including organizing lymphoid structures resembling tertiary lymphoid structures (TLS). Also, analyses of single cell RNA sequencing data of gene expression patterns and signaling pathways reveal a mixed inflammatory microenvironment in BPH. Furthermore, predicted ligand-receptor interactions indicate mixed inflammatory signaling between and among immune cell populations, including T cells, macrophages, and mast cells, that likely to the unresolving nature of BPH inflammation. In all, the results of these studies demonstrate inflammation-induced epithelial and stromal expansion in a mouse model of resolving prostatitis and indicate potential roles for multiple immune cell populations and their interactions in driving the ongoing inflammation of BPH, suggesting that this ongoing inflammation may impact the progressive stromal and epithelial expansion characteristic of BPH

Targeted elastin-like polypeptide fusion protein for near-infrared imaging of human and canine urothelial carcinoma

Cystoscopic visualization of bladder cancer is an essential method for initial bladder cancer detection and diagnosis, transurethral resection, and monitoring for recurrence. We sought to develop a new intravesical imaging agent that is more specific and sensitive using a polypeptide based NIR (near-infrared) probe designed to detect cells bearing epidermal growth factor receptors (EGFR) that are overexpressed in 80% of urothelial carcinoma (UC) cases. The NIR imaging agent consisted of an elastin like polypeptide (ELP) fused with epidermal growth factor (EGF) and conjugated to Cy5.5 to give Cy5.5-N24-EGF as a NIR contrast agent. In addition to evaluation in human cells and tissues, the agent was tested in canine cell lines and tissue samples with naturally occurring invasive UC. Flow cytometry and confocal microscopy were used to test cell-associated fluorescence of the probe in T24 human UC cells, and in K9TCC-SH (high EGFR expression) and K9TCC-Original (low EGF expression) canine cell lines. The probe specifically engages these cells through EGFR within 15 min of incubation and reached saturation within a clinically relevant 1 h timeframe. Furthermore, ex vivo studies with resected canine and human bladder tissues showed minimal signal from normal adjacent tissue and significant NIR fluorescence labeling of tumor tissue, in good agreement with our in vitro findings. Differential expression of EGFR ex vivo was revealed by our probe and confirmed by anti-EGFR immunohistochemical staining. Taken together, our data suggests Cy5.5-ELP-EGF is a NIR probe with improved sensitivity and selectivity towards BC that shows excellent potential for clinical translation