Abnormal expression of bone matrix osteocalcin and sialoprotein by malignant prostate epithelial cells

Rezumat:

Aceasta lucrare este o trecere in revista a datelor din literatura privind bazele moleculare cu referire la expresia anormala a proteinelor matricei osoase, osteocalcina si sialoproteina, de catre celulele epiteliale ale prostatei maligne. Aceste date au relevat faptul ca activitatile de promoter ale osteocalcinei umane si sialoproteinei in linia celulara canceroasa, provenind din cancerul de prostata, androgen –independenta LNCaP, C4-2B, au fost marcant crescute de la 7 la 12 ori intr-o maniera dependenta de concentratie de catre mediul conditionat colectat din cancerul de prostata si celulele strome (b).

Analiza osteocalcinei si regiunile promoter identificate ale sialoproteinei, elemente responsive AMP ciclic(cAMP)-(CRE) arata ca determinanti critici pentru expresia genelor osteocalcinei si sialoproteinei osoase in celulele canceroase ale prostatei. Concluzii: Expresia osteocalcinei si sialoproteinei osoase este coordonata si reglata prin intermediul semnalizarii cAMP dependent PKA care poate defini bazele moleculare ale osteomimetismului, manifestat de catre celulele canceroase ale prostatei.
Cuvinte-cheie: osteocalcina, sialoproteina, cancer de prostata, matrice osoasa

Abstract: 

This paper is a review of literature data on molecular basis regarding the abnormal expression of noncollagenous bone matrix proteins Osteocalcin and sialoprotein by malignant prostate epithelial cells. These data revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b)

Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)–responsive elements (CRE) as the critical determinants for conditioned medium–mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Conclusion: Osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMPdependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.
Keywords: osteocalcin, sialoprotein, prostate cancer, bone matrix, metastasis

Introduction

Despite the prevalence of prostate cancer metastasis to the skeleton, the molecular mechanisms of bone tropism are poorly understood. Previous studies suggest that prostate cancer cell adhesion, extravasation, migration, and interaction with bone cells are critical determinants that govern prostate cancer bone colonization ([1]. Reports using clinical prostate cancer metastasis specimens [2] and experimental cell and animal models [3] found the bone-specific proteins osteocalcin and bone sialoprotein to be expressed in a heterogeneous manner by human prostate cancer specimens.

The authors have proposed that prostate cancer cells acquire osteomimetic or bonelike properties to improve their adhesion, proliferation, and survival in bone [4]. This communication delineates the molecular mechanisms underlying the induction of human osteocalcin and bone sialoprotein promoter activities and their endogenous gene expression in the bone microenvironment by factors secreted from prostate cancer and bone stromal cells.

Because of the potential importance of osteomimicry in enhancing cancer cell adhesion, invasion, and metastasis, the authors have designed studies to define how the expression of bone-specific proteins in prostate cancer cells is regulated. The authors chose to study osteocalcin and bone sialoprotein because of the prevalence of expression of these genes by prostate cancer cell lines [5] and in clinical prostate cancer specimens[6]. Their data showed that osteocalcin and bone sialoprotein expression is stimulated in a selective manner in human prostate cancer but not in bone stromal cell lines.

They observed that the induction of osteocalcin and bone sialoprotein expression in prostate cancer cell lines is mediated by paracrine/ autocrine factors harvested from conditioned medium of prostate cancer and bone stromal cells [7]. The selective nature of soluble factors serving as paracrine mediators in stimulating osteocalcin and bone sialoprotein expression in prostate cancer cell lines agrees with their immunohistochemical staining patterns that are generally heterogeneous [6].

Using the stimulatory response of human osteocalcin/human bone sialoprotein promoter reporter activities and the endogenous steady-state levels of osteocalcin/bone sialoprotein mRNA in C4-2B as the assay end points, the authors showed that the extent of human osteocalcin promoter activation by conditioned medium collected from prostate cancer and bone stromal cell lines correlated directly with the malignant potential of these cells in laboratory immune-compromised mice. The authors also observed that conditioned medium stimulated human osteocalcin and bone sialoprotein promoter activities in a dose-dependent manner and that these increased promoter reporter activities corresponded with the enhancement of the steady-state levels of endogenous osteocalcin and bone sialoprotein mRNA expressed in LNCaP and C4-2B cells.

These results support the idea that osteocalcin and bone sialoprotein, once induced in prostate cancer cells, could facilitate the formation of hydroxyapatite complexes, leading to altered biological functions of prostate cancer cells (i.e., increased cell adhesion, migration and invasion, and recruitment of bone cells). Enhanced bone turnover could contribute to prostate cancer ‘‘seeding’’ in the skeleton.A CRE cis-element, located upstream of the AV region, is responsible for mediating ARCaP conditioned medium activated human osteocalcin promoter activity and osteocalcin mRNA expression in human prostate cell lines . This cis-element seems to be functional in LNCaP and C4-2B cells but remains silent in several other human prostate cancer (DU145, PC3, and ARCaP) and osteosarcoma (MG63) cell lines.

This is supported by the lack of increased osteocalcin expression in these prostate cancer and osteosarcoma cell lines after exposure to ARCaP conditioned medium. Further, nuclear extracts from prostate cancer and bone cells that failed to respond to PKA pathway activators showed a lack of CRE binding and supershift activity. In sharp contrast, however, an increased complex formation between cis-element CRE and trans-acting factor CREB and an expected supershift of this complex were observed in the responsive C4-2B cells on the addition of anti-CREB antibody tion is likely to account for increased human osteocalcin promoter activity and enhanced endogenous osteocalcin mRNA expression in human prostate cancer cells in response to factor(s) from the tumor cell microenvironment.

A similar mechanism regulating human bone sialoprotein promoter activity and mRNA expression was also observed in LNCaP and C4-2B cells after exposure to ARCaP conditioned medium, a result supported by additional studies using interfering pharmacologic agents that either stimulated (db cAMP and forskolin) or repressed (H-89) cAMP accumulation in target cells. Our results show for the first time that conditioned medium harvested from prostate cancer and bone stromal cells stimulated osteocalcin and bone sialoprotein expression primarily through a cAMP-dependent PKA signaling pathway in LNCaP and C4-2B human prostate cancer cells.

This stimulation by conditioned medium lends further support to the observation that osteocalcin and bone sialoprotein are prevalently expressed by clinically localized and bone and lymph node metastatic human prostate cancer tissue specimens and can be the molecular basis of osteomimicry during disease progression.Several growth factors ( fibroblast growth factor-2 or basic fibroblast growth factor, insulin-like growth factor-I, and transforming growth factor-h) and hormones (glucocorticoids, estrogens,parathyroid hormone, and parathyroid hormone–related peptide) have been shown to regulate osteocalcin and bone sialoprotein expression in rodent, chick, or human cell lines.

Boudreaux and Towler[8] showed the induction of osteocalcin promoter activity by a growth factor, fibroblast growth factor-2,in MC3T3-E1 cells. Boguslawski et al.[9] observed that rat and human osteoblast-like cell lines stably transfected with osteocalcin promoter reporter construct responded to parathyroid hormone and growth factors ( fibroblast growth factor-2 and insulin-like growth factor-I) via enhanced osteocalcin transcription mediated by a PKA-dependent pathway. A similar activation of bone sialoprotein promoter activity was documented for fibroblast growth factor-2 [10] parathyroid hormone,and prostaglandin E2 in which bone sialoprotein transcription was stimulated through a PKA-dependent pathway, although a protein kinase C–mediated pathway may play a minor role.

The present study differs from previous reports in two important aspects. First, none of the earlier observations showed conclusively at the molecular level that the involvement of CRE elements within osteocalcin and bone sialoprotein promoters, as defined in the present study, conferred soluble factor–induced osteocalcin and bone sialoprotein expression by cancer or normal cells. Our results emphasized the roles of CRE ciselements in osteomimicry by prostate cancer cells. Second, none of the previously reported factors tested in our assay system activated human osteocalcin and bone sialoprotein promoter activities in C4-2B cells (data not shown). These important distinctions suggest the following possibilities.

(a) There arefundamental differences in terms of the soluble factors, downstream signaling network, and cis-elements within human osteocalcin and bone sialoprotein promoters that are responsible for mediating the osteomimetic properties of prostate cancer and bone cells. (b) At the molecular level, prostate cancer and bone cells may differ in their cell surface receptors and downstream cell signaling pathways responding to soluble factor(s) secreted by prostate cancer and bone stromal cells and their elicited activation of osteocalcin and bone sialoprotein expression. (c) With respect to the osteomimetic response of prostate cancer cells to soluble factors, prostate cancer cell lines clearly show heterogeneity. Because osteocalcin and bone sialoprotein expression in LNCaP, C4-2, and C4-2B cells are responsive to soluble factors and osteocalcin and bone sialoprotein are prevalently expressed in both primary and metastatic human prostate cancer tissues, we suggest that LNCaP and its derivative cell lines are superior models for the study of human prostate cancer progression.

This conclusion is also supported by a large body of literature devoted to the study of the molecular mechanisms of androgen receptor and androgen-independent metastatic [11]. To further understand differences in responsiveness among prostate cancer and bone stromal cells to soluble factors in conditioned medium, it must isolate and characterize the responsible factors and evaluate cell surface or intracellular receptors coupling to cell signaling systems in prostate cancer and bone cells.

In summary, authors data using human prostate cancer cell lines show dramatic cell background–dependent differences in responsiveness to ARCaP conditioned medium and the involvement of the cAMP-PKA signaling cascade in the induction of osteocalcin and bone sialoprotein gene expression in LNCaP and C4-2B cells.

The authors have established at the molecular level that a specific region of ciselement in human osteocalcin promoter, located between _643 and _636 (CRE), must be responsible for conferring cAMP regulation of human osteocalcin promoter activity in human prostate cancer cells. Likewise, other regions of CRE within human bone sialoprotein promoter, _79 to _72 (CRE1) and _674 to _667 (CRE2), must also be activated on exposure of human prostate cancer cells to cAMP mimetics and yet unidentified growth factor(s) in the conditioned medium of prostate cancer and bone stromal cells.

It has been proposed that unknown soluble factors secreted by human prostate cancer or bone stromal cells could assume the key regulatory role in osteocalcin and bone sialoprotein expression in human prostate cancer An ongoing study to identify and characterize the unknown soluble factors in our laboratory has revealed a small polypeptide (<30 kDa) with thermal sensitivity and ammonium sulfate–precipitable characteristics in conditioned medium, which seems exclusively responsible for the activation of human osteocalcin and bone sialoprotein promoter activities in prostate cancer cells. These results could have significant implications for understanding osteomimicry and targeting it therapeutically in human prostate cancer.

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Gabriel Ovidiu Dinu

Floreasca Universitary Emergency Hospital