Lineage plasticity has emerged as an important mechanism of treatment resistance in prostate cancer. Treatmentrefractory prostate cancers are increasingly associated with loss of luminal prostate markers, and in many cases induction of developmental programs, stem cell-like phenotypes, and neuroendocrine/neuronal features. Clinically, lineage plasticity may manifest as low PSA progression, resistance to androgen receptor (AR) pathway inhibitors, and sometimes small cell/neuroendocrine pathologic features observed on metastatic biopsy. This mechanism is not restricted to prostate cancer as other malignancies also demonstrate lineage plasticity during resistance to targeted therapies. At present, there is no established therapeutic approach for patients with advanced prostate cancer developing lineage plasticity or small cell neuroendocrine prostate cancer (NEPC) due to knowledge gaps in the underlying biology. Few clinical trials address questions in this space, and the outlook for patients remains poor. To move forward, urgently needed are: (i) a fundamental understanding of how lineage plasticity occurs and how it can best be defined; (ii) the temporal contribution and cooperation of emerging drivers; (iii) preclinical models that recapitulate biology of the disease and the recognized phenotypes; (iv) identification of therapeutic targets; and (v) novel trial designs dedicated to the entity as it is defined. This Perspective represents a consensus arising from the NCI Workshop on Lineage Plasticity and Androgen Receptor-Independent Prostate Cancer. We focus on the critical questions underlying lineage plasticity and AR-independent prostate cancer, outline knowledge and resource gaps, and identify strategies to facilitate future collaborative clinical translational and basic studies in this space.

Abstract. Nitrogen deposition is projected to increase rapidly in tropical ecosystems, but changes in soil-N-cycling processes in tropical ecosystems under elevated N input are less well understood. We used N-addition experiments to achieve N-enriched conditions in mixedspecies, lowland and montane forests in Panama. Our objectives were to (1) assess changes in soil mineral N production (gross rates of N mineralization and nitrification) and retention (microbial immobilization and rapid reactions to organic N) during 1-and 9-yr N additions in the lowland forest and during 1-yr N addition in the montane forest and (2) relate these changes to N leaching and N-oxide emissions.In the old-growth lowland forest located on an Inceptisol, with high base saturation and net primary production not limited by N, there was no immediate effect of first-year N addition on gross rates of mineral-N production and N-oxide emissions. Changes in soil-N processes were only apparent in chronic (9 yr) N-addition plots: gross N mineralization and nitrification rates, NO 3 À leaching, and N-oxide emissions increased, while microbial biomass and NH 4 þ immobilization rates decreased compared to the control. Increased mineral-N production under chronic N addition was paralleled by increased substrate quality (e.g., reduced C:N ratios of litterfall), while the decrease in microbial biomass was possibly due to an increase in soil acidity. An increase in N losses was reflected in the increase in 15 N signatures of litterfall under chronic N addition.In contrast, the old-growth montane forest located on an Andisol, with low base saturation and aboveground net primary production limited by N, reacted to first-year N addition with increases in gross rates of mineral-N production, microbial biomass, NO 3 À leaching, and Noxide emissions compared to the control. The increased N-oxide emissions were attributed to increased nitrification activity in the organic layer, and the high NO 3 À availability combined with the high rainfall on this sandy loam soil facilitated the instantaneous increase in NO 3 À leaching. These results suggest that soil type, presence of an organic layer, changes in soil-N cycling, and hydrological properties are more important indicators than vegetation as an N sink on how tropical forests respond to elevated N input.

The regulation of epithelial cell function and morphogenesis by the paracrine effectors from the mesenchyme or stroma has been well established using in-vivo studies. A more complete understanding of these relationships has been delayed due, in part, to a lack of appropriate co-culture models. In this study, we describe a co-culture model which demonstrates that normal paracrine relationships can be reconstituted in vitro and that human endometrial stromal cells regulate both growth and differentiation of primary human endometrial epithelial cells. Interesting differences in the proliferation of stromal and epithelial cells were noted in response to the basement membrane extract, Matrigel((R)). Exposure of stromal cells to Matrigel((R)) enhanced the paracrine capacity of these cells in vitro. When epithelial cells were co-cultured in contact with stromal cells embedded in Matrigel((R)), epithelial cell growth was inhibited by 65-80% compared to controls. Stromal cells in contact with Matrigel((R)) also regulated epithelial cell differentiation, as shown by induction of glycodelin expression. These co-culture studies show great promise as a method to investigate the cellular interactions between endometrial stromal and epithelial cells and their environment and to understand the molecular basis for the regulation of normal growth and differentiation of cells within complex tissues such as the endometrium.

The acquisition of an androgen-independent phenotype by prostate cancer cells is presently a death sentence for patients. In order to have a realistic chance of changing this outcome, an understanding of what drives the progression to androgen independence is critical. We review here a working hypothesis based on the position that the development of androgen-independent epithelial cells is the result of a series of cellular and molecular events within the whole tissue that culminates in the loss of normal tissue-maintained growth control. This tissue includes the epithelial and stromal cells, the supporting extracellular matrix and circulating hormones. This review discusses the characteristics of these malignant cells, the role of stromal cells involved in growth and the differentiation of epithelial cells, and the role of the extracellular matrix as a mediator of the phenotypes of stromal and epithelial cells. In addition, environmental, neuroendocrine and immune factors that may contribute to disturbance of the fine balance of the epithelial-stromal-extracellular matrix connection are considered. While the goal of many therapeutic approaches to prostate cancer has been androgen ablation or targeting the androgen receptor (AR) of epithelial cells, these therapies become ineffective as the cells progress beyond dependence on androgen for growth control. Twenty years ago Sir David Smithers debated that cancer is the result of loss of tolerance within tissues and the organizational failure of normal growth-control mechanisms. This is precipitated by prolonged or abnormal demands for regeneration or repair, rather than of any inherent disorder peculiar to each of the individual components involved. He wrote 'It is not the cell itself that is disorderly, but its relationship with the rest of the tissue'. We have gained significantly large amounts of precise data on the effects of androgenic ablation on cancerous prostate cells and on the role of the AR in prostate cancer. The need has come to compile this information towards a perspective of dysregulation of tissue as a whole, and to develop experimental systems to address this broader perspective to find and develop therapies for treatment and prevention.

A panel of expression markers was validated and used to document that, when radical prostatectomy specimens are cultured in low (i.e., <260 Mmol/L)-calcium (Ca 2+ )-serum-free, growth factor-defined (SFD) medium, what grows out are not prostatic cancer cells but basally derived normal transitamplifying prostatic epithelial cells. The selective outgrowth of the normal transit-amplifying versus prostatic cancer cells is due to the differential effect of low-Ca 2+ medium on the structure of Notch-1 and E-cadherin signaling molecules. In low-Ca 2+ medium, Notch-1 receptor is conformationally in a constitutively active, cell autonomous form not requiring reciprocal cell-cell (i.e., ligand) interaction for signaling. Such signaling is required for survival of transit-amplifying cells as shown by the death of transit-amplifying cells induced by treatment with a series of chemically distinct ;-secretase inhibitors to prevent Notch-1 signaling. Conversely, in lowCa 2+ medium, E-cadherin is conformationally inactive preventing cell-cell homotypic interaction, but low cell density nonaggregated transit-amplifying cells still survived because Notch-1 is able to signal cell autonomously. In contrast, when medium Ca 2+ is raised to >400 Mmol/L, Notch-1 conformationally is no longer constitutively active but requires cell-cell contact for reciprocal binding of Jagged-1 ligands and Notch-1 receptors between adjacent transit-amplifying cells to activate their survival signaling. Such cell-cell contact is enhanced by the elevated Ca 2+ inducing an E-cadherin conformation allowing homotypic interaction between transit-amplifying cells. Such Ca 2+ -dependent, E-cadherin-mediated interaction, however, results in cell aggregation, stratification, and inhibition of proliferation of transit-amplifying cells via contact inhibition-induced up-regulation of p27/kip1 protein.In addition, transit-amplifying cells not contacting other cells undergo squamous differentiation into cornified (i.e., 1% SDS insoluble) envelopes and death in the elevated Ca 2+ medium. Stratification and contact inhibition induced by elevated Ca 2+ are dependent on E-cadherin-mediated homotypic interaction between transit-amplifying cells as shown by their prevention in the presence of a cell-impermanent, E-cadherin neutralizing antibody. In contrast to growth inhibition of normal transit-amplifying cells, supplementation of low-Ca 2+ -SFD medium with 10% FCS and raising the Ca 2+ to >600 Mmol/L stimulates the growth of all prostate cancer cell lines tested.Additional results document that, at physiologic levels of Ca 2+ (i.e., >600 Mmol/L), prostatic cancer cells are not contact inhibited by E-cadherin interactions and Notch-1 signaling is no longer required for survival but instead becomes one of multiple signaling pathways for proliferation of prostatic cancer cells. These characteristic changes are consistent with prostate cancer cells' ability to metastasize to bone, a site of high-Ca 2+ levels. (Cancer Res 2005; 65(20): 9269-79)

BACKGROUND Prostatic cancer cells are lethal because they acquire the ability to activate survival pathways that do not require androgenic stimulation. As a rational approach to developing effective therapy for these devastating cells, specific signal transduction pathways uniquely required for the survival of these nonandrogen‐dependent prostate cancer cells must be identified. Previous studies suggested that the neurotrophin/trk signal transduction axis may regulate such unique survival pathways. In the present study, the changes in expression of the neurotrophins (NGF, BDNF, and NT‐3) and their cognate receptors (i.e., trk and p75NTR) during the progression of normal prostatic epithelial cells to malignancy were documented. Additionally, the consequences of inhibiting these trk signaling pathways on the in vitro survival of prostate cancer cells was tested. METHODS Immmunocytochemistry, RT‐PCR, and ELISA assays were used to characterize the changes in the neurotrophin ligands (i.e., NGF, BDNF, and NT‐3) and their cognate high‐affinity (i.e., trk A, B, and C) and low‐affinity neurotrophin (i.e., p75 NTR) receptors in normal vs. malignant human prostatic tissues. CEP‐751 is an indolocarbazole compound specifically designed to inhibit the initiation of these neurotrophin/trk signal transductions. The consequence of CEP‐751 inhibition of trk signaling for in vitro clonogenic survival of a series of human prostatic cancer lines was also tested. RESULTS These studies demonstrated that normal prostatic tissue from patients without prostate cancer contains substantial levels of nerve growth factor (NGF), which is produced in a paracrine manner by stromal cells. These stromal cells lack both trk and p75NTR receptors. In contrast, normal prostatic epithelial cells from patients without prostate cancer do not secrete detectable levels of neurotrophins, but do express trk A and p75 NTR. While the NGF/trkA/p75 NTR axis is present in the normal prostate, normal prostatic epithelial cells do not depend on this axis for their survival. In contrast, malignant prostate epithelial cells directly secrete a series of neurotrophins (i.e., NGF, BDNF, and/or NT‐3) and express at least one if not more of the trk receptor proteins (i.e., trk A, B, and/or C), while no longer expressing the p75NTR receptors. In addition, inhibition of autocrine trk signaling via CEP‐751 treatment induces the apoptotic death of these malignant cells. CONCLUSIONS Prostate carcinogenesis involves molecular changes leading to the paracrine and/or autocrine production of a series of neurotrophins. This is coupled to the ectopic expression of trk B and trk C, as well as to the continued expression of trk A, and the loss of expression of p75NTR receptors. These changes result in the acquisition by malignant prostate cells of a unique requirement for trk signaling pathways for survival. Based on these findings, trk inhibition is a novel, rational approach for prostate cancer therapy. Prostate 45:140–148, 2000. © 2000 Wiley‐Liss, Inc.

Comparative effects of DHEA vs. testosterone, dihydrotestosterone, and estradiol on proliferation and gene expression in human LNCaP prostate cancer cells.

No abstract