Johng S. Rhim

ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.

We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway

Interactions between Eph receptor tyrosine kinases (RTKs) and membrane-anchored ephrin ligands critically regulate axon pathfinding and development of the cardiovascular system, as well as migration of neural cells. Similar to other RTKs, ligand-activated Eph kinases recruit multiple signalling and adaptor proteins, several of which are involved in growth regulation. However, in contrast to other RTKs, activation of Eph receptors fails to promote cell proliferation or to transform rodent fibroblasts, indicating that Eph kinases may initiate signalling pathways that are distinct from those transmitted by other RTKs. Here we show that stimulation of endogenous EphA kinases with ephrin-A1 potently inhibits the Ras/MAPK cascade in a range of cell types, and attenuates activation of mitogen-activated protein kinase (MAPK) by receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). In prostatic epithelial cells and endothelial cells, but not fibroblasts, treatment with ephrin-A1 inhibits cell proliferation. Our results identify EphA kinases as negative regulators of the Ras/MAPK pathway that exert anti-mitogenic functions in a cell-type-specific manner.

Overexpression of the EphA2 tyrosine kinase in prostate cancer

Molecules that are highly expressed by human prostate cancers may serve as therapeutically relevant targets or tumor markers. Tyrosine kinases are frequently overexpressed in metastatic tumor cells and this prompted us to screen for tyrosine kinases that are overexpressed in prostate cancer cells.

Duffy Antigen Facilitates Movement of Chemokine Across the Endothelium In Vitro and Promotes Neutrophil Transmigration In Vitro and In Vivo

The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-α/CXC chemokine ligand 1 (GRO-α/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-α/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-α/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by β1 integrins

We show here that β1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The β1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, β1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, β1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the β1 cytodomain plays an important role in mediating β1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, β1A associates with IRS-1 and β1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.

OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation.

Background Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common in multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction. Methodology/Principal Findings Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -independent growth of carcinoma cells with endogenous silencing. Conclusions/Significance Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies.

Regulation of HMGA1 Expression by MicroRNA-296 Affects Prostate Cancer Growth and Invasion

Purpose: High-motility group AT-hook gene 1 (HMGA1) is a non-histone nuclear binding protein that is developmentally regulated. HMGA1 is significantly overexpressed in and associated with high grade and advance stage of prostate cancer (PC). The oncogenic role of HMGA1 is at least mediated through chromosomal instability and structural aberrations. However, regulation of HMGA1 expression is not well understood. Identification of microRNA-mediated HMGA1 regulation will provide a promising therapeutic target in treating PC. Experimental Design: In this study, we examined the functional relation between miR-296 and HMGA1 expression in several PC cell lines and a large PC cohort. We further examined the oncogenic property of HMGA1 regulated by miR-296. Results: Here we report that miR-296, a microRNA predicted to target HMGA1, specifically represses HMGA1 expression by promoting degradation and inhibiting HMGA1translation. Repression of HMGA1 by miR-296 is direct and sequence specific. Importantly, ectopic miR-296 expression significantly reduced PC cell proliferation and invasion, in part through the downregulation of HMGA1. Examining PC patient samples, we found an inverse correlation between HMGA1 and miR-296 expression: high levels of HMGA1 were associated with low miR-296 expression and strongly linked to more advanced tumor grade and stage. Conclusions: Our results indicate that miR-296 regulates HMGA1 expression and is associated with PC growth and invasion. Clin Cancer Res; 17(6); 1297–305. ©2010 AACR.

Antisense to the Early Growth Response-1 Gene (Egr-1) Inhibits Prostate Tumor Development in TRAMP Mice

Abstract: Egr‐1 is a transcription factor induced by stress or injury, mitogens, and differentiation factors. Egr‐1 regulates the expression of genes involved in growth control or survival. Expression of Egr‐1 results in either promotion or regression of cell proliferation, depending on cell type and environment. Egr‐1 acts as a tumor suppressor in many cell types and loss of Egr‐1 has been proposed to contribute to cancer progression. There is strong new evidence however suggesting that Egr‐1 overexpression is involved in prostate cancer progression. For example, Egr‐1 expression levels are elevated in human prostate carcinomas in proportion to grade and stage. Furthermore, prostate cancer progression was significantly delayed in two models of prostate cancer mice lacking Egr‐1. Our objective in the present study is to test whether inhibition of Egr‐1 function would block cell proliferation and inhibit the transformed phenotype of prostate cancer cells in vitro and in vivo. We describe the development of high affinity and high specificity antisense oligonucleotides that efficiently inhibit Egr‐1 expression. We show that inhibition of Egr‐1 expression in mouse or human prostate cancer cells decreased proliferation and reduced the capacity of these cells to form colonies and to grow in soft agar. Conversely, stable expression of Egr‐1 in normal human prostate epithelial 267B1 cells promoted transformation. In TRAMP mice, treatment with Egr‐1 antisense oligonucleotides delayed the occurrence of prostate tumors. Importantly, Egr‐1 antisense showed little or no toxicity when injected into animals. Finally, we identified a few genes such as cyclin D2, p19ink4d, and Fas that are directly regulated by Egr‐1 in prostate cancer cells and that control cell cycle and survival.

Immortalization of nasal polyp epithelial cells from cystic fibrosis patients

We have developed immortalized epithelial cystic fibrosis (CF) cell lines by infecting cultured nasal polyp cells with a SV40/Adenol2 hybrid virus. The cell lines obtained are epithelial in nature as shown by cytokeratin production and morphology, although cytokeratins 4 and 13 typical of primary nasal polyp cells are produced at a much reduced rate. Ussing chamber experiments showed that the precrisis CF cell line NCF3 was able to perform trans-cellular chloride transport when activated by agents which elevate intracellular calcium. cAMP agonists had no effect on chloride flux in NCF3 as expected for CF cells. The apical chloride channels found with the patch clamp technique in NCF3 and in the postcrisis cell line NCF3A have a conductance similar to that of chloride channels found earlier in normal and CF epithelial cells. The channels show a delay in the onset of activity in off-cell patches and are not activated by increased cAMP levels in the cell. This indicates that immortalized CF epithelial cells will provide a useful model for the study of cystic fibrosis.

HPMA copolymer-based combination therapy toxic to both prostate cancer stem/progenitor cells and differentiated cells induces durable anti-tumor effects

Current treatments for prostate cancer are still not satisfactory, often resulting in tumor regrowth and metastasis. One of the main reasons for the ineffective anti-prostate cancer treatments is the failure to deplete cancer stem-like cells (CSCs) - a subset of cancer cells with enhanced tumorigenic capacity. Thus, combination of agents against both CSCs and bulk tumor cells may offer better therapeutic benefits. Several molecules with anti-cancer stem/progenitor cell activities have been under preclinical evaluations. However, their low solubility and nonspecific toxicity limit their clinical translation. Herein, we designed a combination macromolecular therapy containing two drug conjugates: HPMA copolymer-cyclopamine conjugate (P-CYP) preferentially toxic to cancer stem/progenitor cells, and HPMA copolymer-docetaxel conjugate (P-DTX) effective in debulking the tumor mass. Both conjugates were synthesized using RAFT (reversible addition-fragmentation chain transfer) polymerization resulting in narrow molecular weight distribution. The killing effects of the two conjugates against bulk tumor cells and CSCs were evaluated in vitro and in vivo. In PC-3 or RC-92a/hTERT prostate cancer cells, P-CYP preferentially kills and impairs the function of CD133+ prostate cancer stem/progenitor cells; P-DTX was able to kill bulk tumor cells instead of CSCs. In a PC-3 xenograft mice model, combination of P-DTX and P-CYP showed the most effective and persistent tumor growth inhibitory effect. In addition, residual tumors contained less CD133+ cancer cells following combination or P-CYP treatments, indicating selective killing of cancer cells with stem/progenitor cell properties.