Robin Schneider-Broussard

Highly purified CD44 + prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells

CD44 is a multifunctional protein involved in cell adhesion and signaling. The role of CD44 in prostate cancer (PCa) development and progression is controversial with studies showing both tumor-promoting and tumor-inhibiting effects. Most of these studies have used bulk-cultured PCa cells or PCa tissues to carry out correlative or overexpression experiments. The key experiment using prospectively purified cells has not been carried out. Here we use FACS to obtain homogeneous CD44+ and CD44− tumor cell populations from multiple PCa cell cultures as well as four xenograft tumors to compare their in vitro and in vivo tumor-associated properties. Our results reveal that the CD44+ PCa cells are more proliferative, clonogenic, tumorigenic, and metastatic than the isogenic CD44− PCa cells. Subsequent molecular studies demonstrate that the CD44+ PCa cells possess certain intrinsic properties of progenitor cells. First, BrdU pulse-chase experiments reveal that CD44+ cells colocalize with a population of intermediate label-retaining cells. Second, CD44+ PCa cells express higher mRNA levels of several ‘stemness’ genes including Oct-3/4, Bmi, β-catenin, and SMO. Third, CD44+ PCa cells can generate CD44− cells in vitro and in vivo. Fourth, CD44+ PCa cells, which are AR−, can differentiate into AR+ tumor cells. Finally, a very small percentage of CD44+ PCa cells appear to undergo asymmetric cell division in clonal analyses. Altogether, our results suggest that the CD44+ PCa cell population is enriched in tumorigenic and metastatic progenitor cells.

Hierarchical organization of prostate cancer cells in xenograft tumors: The CD44+α2β1+ cell population is enriched in tumor-initiating cells

Prostate cancer cells are heterogeneous in their tumorigenicity. For example, the side population cells isolated from LAPC9 xenografts are 100 to 1,000 times more tumorigenic than the corresponding non–side population cells. Highly purified CD44 + prostate cancer cells from several xenografts are also enriched in prostate cancer stem/progenitor cells. Because the CD44 + prostate cancer cell population is still heterogeneous, we wonder whether we could further enrich for tumorigenic prostate cancer cells in this population using other markers. Integrin A2B1 has been proposed to mark a population of normal human prostate stem cells. Therefore, we first asked whether the A2B1 +/hi cells in prostate tumors might also represent prostate cancer stem cells. Highly purified (z98%) A2B1 +/hi cells from three human xenograft tumors, Du145, LAPC4, and LAPC9, show higher clonal and clonogenic potential than the A2B1 � /lo cells in vitro. However, when injected into the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse prostate or s.c., the A2B1 +/hi prostate cancer cells are no more tumorigenic than the A2B1 � /lo cells. Immunofluorescence studies reveal that CD44 and A2B1 identify an overlapping and inclusive population of prostate cancer cells in that f70% of A2B1 +/hi cells are CD44 + and 20% to 30% of CD44 + cells are distributed in the A2B1 � /lo cell population. Subsequently, we sorted out CD44 + A2B1 +/hi ,C D44 + A2B1 � /lo ,C D44 � A2B1 +/hi ,a nd CD44 � A2B1 � /lo cells from LAPC9 tumors and carried out tumorigenicity experiments. The results revealed a hierarchy in tumorigenic potential in the order of CD44 + A2B1 +/hi � CD44 + A2B1 � /lo > CD44 � A2B1 +/hi J CD44 � A2B1 � /lo . These observations together suggest that prostate cancer cells are organized as a hierarchy. [Cancer Res 2007;67(14):6796–805]

Prostate cancer stem/progenitor cells: Identification, characterization, and implications

Several solid tumors have now been shown to contain stem cell‐like cells called cancer stem cells (CSC). These cells, although generally rare, appear to be highly tumorigenic and may be the cells that drive tumor formation, maintain tumor homeostasis, and mediate tumor metastasis. In this Perspective, we first provide our insight on how a CSC should be defined. We then summarize our current knowledge of stem/progenitor cells in the normal human prostate (NHP), an organ highly susceptible to hyperproliferative diseases such as benign prostate hyperplasia (BPH) and prostate cancer (PCa). We further review the evidence that cultured PCa cells, xenograft prostate tumors, and patient tumors may contain stem/progenitor cells. Along with our discussion, we present several methodologies that can be potentially used to identify putative tumor‐reinitiating CSC. Finally, we present a hypothetical model for the hierarchical organization of human PCa cells and discuss the implications of this model in helping understand prostate carcinogenesis and design novel diagnostic, prognostic, and therapeutic approaches.

E2F1 Has Both Oncogenic and Tumor-Suppressive Properties in a Transgenic Model

ABSTRACT Using a transgenic mouse model expressing the E2F1 gene under the control of a keratin 5 (K5) promoter, we previously demonstrated that increased E2F1 activity can promote tumorigenesis by cooperating with either a v-Ha-ras transgene to induce benign skin papillomas or p53 deficiency to induce spontaneous skin carcinomas. We now report that as K5 E2F1 transgenic mice age, they are predisposed to develop spontaneous tumors in a variety of K5-expressing tissues, including the skin, vagina, forestomach, and odontogenic epithelium. On the other hand, K5 E2F1 transgenic mice are found to be resistant to skin tumor development following a two-stage carcinogenesis protocol. Additional experiments suggest that this tumor-suppressive effect of E2F1 occurs at the promotion stage and may involve the induction of apoptosis. These findings demonstrate that increased E2F1 activity can either promote or inhibit tumorigenesis, dependent upon the experimental context.

Regulation of BRCA1 expression by the Rb-E2F pathway.

Inheritance of a mutant allele of the breast cancer susceptibility gene BRCA1 confers increased risk of developing breast and ovarian cancers. Likewise, inheritance of a mutant allele of the retinoblastoma susceptibility gene (RB1) results in the development of retinoblastoma and/or osteosarcoma, and both alleles are often mutated or inactivated in sporadic forms of these and other cancers. We now demonstrate that the product of the RB1 gene, Rb, regulates the expression of the murine Brca1 and human BRCA1 genes through its ability to modulate E2F transcriptional activity. TheBrca1 gene is identified as an in vivo target of E2F1 in a transgenic mouse model. The Brca1 promoter contains E2F DNA-binding sites that mediate transcriptional activation by E2F1 and repression by Rb. Moreover, ectopic expression of cyclin D1 and Cdk4 can stimulate the Brca1 promoter in an E2F-dependent manner, and this is inhibited by coexpression of the p16 INK4a cyclin-dependent kinase inhibitor. The human BRCA1 promoter also contains a conserved E2F site and is similarly regulated by E2F1 and Rb. This functional link between the BRCA1 and Rb tumor suppressors may provide insight into the mechanism by which BRCA1 inactivation contributes to cancer development.

Myc lacks E2F1's ability to suppress skin carcinogenesis.

Myc and E2F1 can each stimulate proliferation, induce apoptosis, and contribute to oncogenic transformation. However, only E2F1 has been shown to have a tumor suppressive activity under some conditions. To examine the potential of Myc to suppress tumorigenesis under one of the conditions in which E2F1 functions to suppress tumorigenesis, transgenic mice expressing Myc under the control of a keratin 5 (K5) promoter were generated. Like K5 E2F1 transgenic mice, K5 Myc transgenic mice have hyperplastic and hyperproliferative epidermis and develop spontaneous tumors in the skin and oral epithelium. In addition, K5 Myc and K5 E2F1 transgenic mice both display aberrant, p53-dependent apoptosis in the epidermis. It has been demonstrated that deregulated expression of E2F1 in the epidermis of transgenic mice inhibits tumorigenesis in a two-stage skin carcinogenesis assay. In sharp contrast to those results, deregulated expression of Myc in the epidermis of transgenic mice resulted in an enhanced response to two-stage skin carcinogenesis. We conclude that while Myc and E2F1 have similar proliferative, apoptotic and oncogenic properties in mouse epidermis, Myc lacks E2F1s tumor suppressive property. This suggests that E2F1s unique ability to inhibit skin carcinogenesis is not simply a consequence of promoting p53-dependent apoptosis.

Differential activities of E2f family members: Unique functions in regulating transcription

Several regulators of E2F transcriptional activity, including the retinoblastoma tumor suppressor (Rb) protein, p16Ink4a, cyclin D1, and cyclin‐dependent kinase 4, have been shown to be targets for genetic alterations that underlie the development of human cancers. Deregulation of E2F transcription factors as a result of these genetic alterations is believed to contribute to tumor development. This hypothesis is supported by the finding that at least some members of the E2F gene family can contribute to oncogenic transformation when overexpressed. Each E2F family member can dimerize with DP proteins, bind consensus E2F sites, and activate transcription. Several pieces of evidence suggest, however, that the various E2F species have unique functions in regulating transcription. We compared the abilities of E2F1, E2F4, and E2F5 to activate transcription from a variety of gene promoters and found that in all cases E2F1 was the most potent activator, followed by E2F4 and then by E2F5. Construction of chimeric proteins between E2F1 and E2F4 demonstrated that either the carboxy terminus or the amino terminus of E2F1 could make E2F4 a more potent activator. In contrast, neither the carboxy terminus nor the amino terminus of E2F1 could significantly increase the activity of E2F5. We found that, consistent with a role for E2F5 in transcriptional repression, E2F5s binding partner p130, like Rb, could also actively repress transcription when directly bound to a target promoter. Mol. Carcinog. 22:190–198, 1998.

Critical and Distinct Roles of p16 and Telomerase in Regulating the Proliferative Life Span of Normal Human Prostate Epithelial Progenitor Cells

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, α2β1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.

Evidence that senescent human prostate epithelial cells enhance tumorigenicity: Cell fusion as a potential mechanism and inhibition by p16INK4a and hTERT

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo but the potential role of senescent NHP cells in prostate tumorigenesis remain unclear. Here we show that senescent NHP cells enhance the in vivo tumorigenicity of low‐tumorigenic LNCaP prostate cancer and low/non‐tumorigenic subset of cells (called L cells) isolated from multiple bulk‐cultured prostate (and other) cancer cell lines. Subsequent studies suggest cell–cell fusion as a potential mechanism for senescent NHP cell‐enhanced tumor development. Using fluorescently tagged tumor cells and/or NHP cells, we find that NHP cells, like fibroblasts, can undergo fusion with unfractionated tumor cells or the L cells. Using 293T‐L cells as the model cell system, we verify NHP and 293T‐L cell fusion by using differential RT‐PCR, karyotyping, and gene expression analyses. Further experiments demonstrate that senescent NHP cells that have lost progenitor markers, accumulated p16INK4a (p16) protein expression, and acquired the AR mRNA expression, appear to be the preferential fusion targets. Strikingly, the tumorigenicity of the NHP/293T‐L hybrid cells was inhibited by exogenous p16 as well as hTERT. Chromosomal analyses revealed that hTERT probably inhibited the in vivo tumorigenicity by maintaining genomic stability. These results suggest that senescent NHP cells, like senescent fibroblasts, may promote tumor development and that one of the mechanisms underlying the senescent NHP cell‐enhanced tumorigenicity could be through cell fusion.

TRANSGENIC EXPRESSION OF 15-LIPOXYGENASE 2 (15-LOX2) IN MOUSE PROSTATE LEADS TO HYPERPLASIA AND CELL SENESCENCE

15-Lipoxygenase 2 (15-LOX2), a lipid-peroxidizing enzyme, is mainly expressed in the luminal compartment of the normal human prostate, and is often decreased or lost in prostate cancer. Previous studies from our lab implicate 15-LOX2 as a functional tumor suppressor. To better understand the biological role of 15-LOX2 in vivo, we generated prostate-specific 15-LOX2 transgenic mice using the ARR2PB promoter. Unexpectedly, transgenic expression of 15-LOX2 or 15-LOX2sv-b, a splice variant that lacks arachidonic acid-metabolizing activity, resulted in age-dependent prostatic hyperplasia and enlargement of the prostate. Prostatic hyperplasia induced by both 15-LOX2 and 15-LOX2sv-b was associated with an increase in luminal and Ki-67+ cells; however, 15-LOX2-transgenic prostates also showed a prominent increase in basal cells. Microarray analysis revealed distinct gene expression profiles that could help explain the prostate phenotypes. Strikingly, 15-LOX2, but not 15-LOX2sv-b, transgenic prostate showed upregulation of several well-known stem or progenitor cell molecules including Sca-1, Trop2, p63, Nkx3.1 and Psca. Prostatic hyperplasia caused by both 15-LOX2 and 15-LOX2sv-b did not progress to prostatic intraprostate neoplasia or carcinoma and, mechanistically, prostate lobes (especially those of 15-LOX2 mice) showed a dramatic increase in senescent cells as revealed by increased SA-βgal, p27Kip1 and heterochromatin protein 1γ staining. Collectively, our results suggest that 15-LOX2 expression in mouse prostate leads to hyperplasia and also induces cell senescence, which may, in turn, function as a barrier to tumor development.