pRb inactivation in mammary cells reveals common mechanisms for tumor initiation and progression in divergent epithelia

Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb_f, collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb_f loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb_f function; Rb_f inactivation by T₁₂₁, a fragment of SV40 T antigen that binds to and inactivates pRb_f proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.     

Dendritic cells in colorectal cancer correlate with other tumor-infiltrating immune cells

Dendritic cells (DCs) are the most efficient antigen-presenting cells and play a key role in a cellular antitumor immune response. In this study we investigated the exact localization of DCs within colorectal tumors and their relationship to tumor-infiltrating lymphocytes as well as clinical outcome of the patients. Primary tumor specimens of 104 patients with a diagnosis of colorectal cancer were identified retrospectively and analyzed with the dendritic cell markers S-100 protein and human leukocyte antigens (HLA) class II. The markers were individually combined with laminin as a second marker to facilitate the observation of the different tumor localizations. S-100 or HLA class II positive cells were found in the three different compartments of colorectal tumors: tumor epithelium, tumor stroma, and advancing tumor margin, but mainly present in tumor stroma and advancing tumor margin. S-100-positive tumor-infiltrating DCs in direct contact with tumor cells, i.e., in tumor epithelium, significantly correlated to the intraepithelial infiltration of CD4+ (p=0.02) and CD8+ (p=0.01) lymphocytes. High HLA class II+ cell infiltration in the tumor stroma correlated to a lower intraepithelial infiltration of CD8+ (p=0.02) lymphocytes. High intraepithelial infiltration of S-100-positive DCs suggested increased disease-free survival, but was not statistically significant, while high amounts of HLA class II+ cells in the tumor stroma correlated with an adverse survival outcome. Our results show that the infiltration of DCs in colorectal cancer, depending on both location and type of marker, is correlated with local immune interactions and patient prognosis, suggesting a central role for DCs in controlling local tumor immunity.     

Development of a facile fluorescent assay for the detection of 80 mutations within the p53 gene

BACKGROUND: Alterations in the p53 tumor suppressor gene constitute one of the most frequent genetic events associated with the development of human cancers. Determination of an individual's p53 status may be of value in early diagnosis, prediction of response to treatment, and for the detection of minimal residual cancer. Recent studies have also revealed that specific mutations affecting the p53 gene are associated with a poor outcome. The majority of tumor biopsies that are sent for study in the laboratory contain neoplastic cells intermingled with stroma, such that the detection of alterations in the p53 gene requires a tumor enrichment technique and/or highly sensitive mutation detection technologies. Thus, it is desirable that a clinically useful assay for detecting point mutations in the p53 gene function in the presence of significant quantities of wild-type sequence and identify the critical sequence aberrations. MATERIALS AND METHODS: We utilized molecular beacons in a real-time allele-specific PCR format to obtain reference data on samples of quantitatively known p53 mutation status. These data have been statistically analyzed and the results used to detect p53 mutations, indicating the presence of occult tumor. RESULTS: We describe validation of a simple, rapid, sensitive, and quantitative ARMS assay for identifying the levels of 80 point mutations within the p53 gene that, when mutated, constitute at least 1% of the total p53 sequences. CONCLUSIONS: The assay successfully identifies rare p53 gene mutations in clinical samples and overcomes many of the limitations of current technologies.     

Loss of heterozygosity and microsatellite instability in tumor-associated stromal cells and tumor epithelium of prostate cancer

In the past decade, intense studies of the tumor microenvironment yielded ample data testifying to the critical role of stroma in carcinogenesis. Genetic lesions accumulate not only in the tumor epithelium, but also in tumor-associated fibroblasts. The epithelial and stromal components of prostate cancer (PC) and prostatic intraepithelial neoplasia (PIN) were isolated by laser capture microdissection and subjected to microsatellite analysis of chromosome regions 8p22, 13q14, and 16q23. The frequency of allele alterations in the epithelium was 48% for 8p22, 72% for 13q14, and 37% for 16q23. Slightly higher frequencies of the loss of heterozygosity (LOH) and microsatellite instability in these loci were observed in tumor-associated stroma. Molecular alterations were also found in both epithelial (16–27%) and stromal (8–22%) components in PIN. LOH on chromosomes 16 and 13 in the epithelium was significantly associated with the Gleason score, PC stage, and metastasis into regional lymph nodes. Thus, multiple genetic aberrations occur in the stromal component of PC as frequently as in the tumor epithelium.     

Involvement of stromal p53 in tumor-stroma interactions

p53 is a major tumor-suppressor gene, inactivated by mutations in about half of all human cancer cases, and probably incapacitated by other means in most other cases. Most research regarding the role of p53 in cancer has focused on its ability to elicit apoptosis or growth arrest of cells that are prone to become malignant owing to DNA damage or oncogene activation, i.e. cell-autonomous activities of p53. However, p53 activation within a cell can also exert a variety of effects upon neighboring cells, through secreted factors and paracrine and endocrine mechanisms. Of note, p53 within cancer stromal cells can inhibit tumor growth and malignant progression. Cancer cells that evolve under this inhibitory influence acquire mechanisms to silence stromal p53, either by direct inhibition of p53 within stromal cells, or through pressure for selection of stromal cells with compromised p53 function. Hence, activation of stromal p53 by chemotherapy or radiotherapy might be part of the mechanisms by which these treatments cause cancer regression. However, in certain circumstances, activation of stromal p53 by cytotoxic anti-cancer agents might actually promote treatment resistance, probably through stromal p53-mediated growth arrest of the cancer cells or through protection of the tumor vasculature. Better understanding of the underlying molecular mechanisms is thus required. Hopefully, this will allow their manipulation towards better inhibition of cancer initiation, progression and metastasis.     

Irradiation Selects for p53-Deficient Hematopoietic Progenitors

Identification and characterization of mutations that drive cancer evolution constitute a major focus of cancer research. Consequently, dominant paradigms attribute the tumorigenic effects of carcinogens in general and ionizing radiation in particular to their direct mutagenic action on genetic loci encoding oncogenes and tumor suppressor genes. However, the effects of irradiation are not limited to genetic loci that encode oncogenes and tumor suppressors, as irradiation induces a multitude of other changes both in the cells and their microenvironment which could potentially affect the selective effects of some oncogenic mutations. P53 is a key tumor suppressor, the loss of which can provide resistance to multiple genotoxic stimuli, including irradiation. Given that p53 null animals develop T-cell lymphomas with high penetrance and that irradiation dramatically accelerates lymphoma development in p53 heterozygous mice, we hypothesized that increased selection for p53-deficient cells contributes to the causal link between irradiation and induction of lymphoid malignancies. We sought to determine whether ionizing irradiation selects for p53-deficient hematopoietic progenitors in vivo using mouse models. We found that p53 disruption does not provide a clear selective advantage within an unstressed hematopoietic system or in previously irradiated BM allowed to recover from irradiation. In contrast, upon irradiation p53 disruption confers a dramatic selective advantage, leading to long-term expansion of p53-deficient clones and to increased lymphoma development. Selection for cells with disrupted p53 appears to be attributable to several factors: protection from acute irradiation-induced ablation of progenitor cells, prevention of irradiation-induced loss of clonogenic capacity for stem and progenitor cells, improved long-term maintenance of progenitor cell fitness, and the disabling/elimination of competing p53 wild-type progenitors. These studies indicate that the carcinogenic effect of ionizing irradiation can in part be explained by increased selection for cells with p53 disruption, which protects progenitor cells both from immediate elimination and from long-term reductions in fitness following irradiation.     

Mesenchymal-epithelial interactions: past, present, and future

Abstract This review summarizes the history of research on mesenchymal–epithelial interactions in prostatic development from the first studies in 1970 to the present. From this study we have learned that prostatic development requires a reciprocal interaction between epithelium and mesenchyme in which urogenital sinus mesenchyme induces and patterns epithelial development and differentiation, while developing prostatic epithelium induces and patterns mesenchymal differentiation into smooth muscle and other resident cell types in the stroma. Prostatic development requires androgen action mediated by the androgen receptor (AR). Through analysis of tissue recombinants composed of wild-type and AR-null epithelium and mesenchyme, we have learned that many "androgenic effects" on prostatic epithelium do not require epithelial AR, but instead are elicited by the paracrine action of AR-positive mesenchyme. Present and future studies reviewed in this issue deal with the molecular mechanisms in this developmental communication between epithelium and mesenchyme.     

Atm is dispensable for p53 apoptosis and tumor suppression triggered by cell cycle dysfunction

Both p53 and ATM are checkpoint regulators with roles in genetic stabilization and cancer susceptibility. ATM appears to function in the same DNA damage checkpoint pathway as p53. However, ATM's role in p53-dependent apoptosis and tumor suppression in response to cell cycle dysregulation is unknown. In this study, we tested the role of murine ataxia telangiectasia protein (Atm) in a transgenic mouse brain tumor model in which p53-mediated apoptosis results in tumor suppression. These p53-mediated activities are induced by tissue-specific inactivation of pRb family proteins by a truncated simian virus 40 large T antigen in brain epithelium. We show that p53-dependent apoptosis, transactivation, and tumor suppression are unaffected by Atm deficiency, suggesting that signaling in the DNA damage pathway is distinct from that in the oncogene-induced pathway. In addition, we show that Atm deficiency has no overall effect on tumor growth and progression in this model.     

Aberrant overexpression of 53BP2 mRNA in lung cancer cell lines

The p53-binding protein 2 (53BP2) was identified as a binding protein to a tumor suppressor p53. We examined the genetic aberrations of 53BP2 gene in various human cancer cell lines. Although no gross genomic alteration or mutation of 53BP2 gene was observed, 53BP2 mRNA levels were highly variable. There was no association between the 53BP2 mRNA level and the p53 status. When we examined sensitivities of these cell lines to DNA-damaging agents including UV irradiation, X-ray irradiation and cis-diamine-dichloroplatinum (CDDP), we found that higher 53BP2 mRNA expression was correlated with the sensitivity to these agents.     

The germ layer origin of mouse vaginal epithelium restricts its responsiveness to mesenchymal inductors: uterine induction

The epithelium of the mammalian vagina arises from two distinct germ layers, endoderm from the urogenital sinus and mesoderm from the lower fused Müllerian ducts. While previously it has been reported that neonatal vaginal epithelium can be induced to differentiate as uterus, which normally develops from the middle portion of the Müllerian ducts, it has not been determined whether this ability is shared by both mesoderm- and endoderm-derived vaginal epithelia. To test if germ layer origin influences the ability of vaginal epithelium to undergo uterine differentiation, we have isolated sinus-derived and Müllerian-derived vaginal epithelia from newborn mice, combined them with uterine mesenchyme, and grown them for 4 weeks in female mice. Mesoderm-derived Müllerian vaginal epithelium in combination with uterine mesenchyme formed the simple columnar epithelium typical of uterus. Similar results were obtained with neonatal cervical epithelium, another mesodermal Müllerian duct derivative. On the other hand, sinus vaginal epithelium combined with uterine mesenchyme formed small cysts lined by a stratified squamous vaginal-like epithelium. This epithelium never showed evidence of cycling between the cornified and mucified states as is typically seen in vaginal epithelium combined with vaginal stroma. These results indicate that the ability of epithelium to form uterus is limited to mesoderm-derived epithelia and suggest that endoderm-derived sinus vaginal epithelium cannot undergo the typical differentiative modifications in response to the hormonal fluctuations of the estrous cycle when associated with uterine stroma.     

Stromal biology of pancreatic cancer

The genetic paradigm of cancer, focused largely on sequential molecular aberrations and associated biological impact in the neoplastic cell compartment of malignant tumors, has dominated our view of cancer pathogenesis. For the most part, this conceptualization has overlooked the dynamic and complex contributions of the surrounding microenvironment comprised of non-tumor cells (stroma) that may resist, react to, and/or foster tumor development. Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease in which a prominent tumor stroma compartment is a defining characteristic. Indeed, the bulk of PDAC tumor volume consists of non-neoplastic fibroblastic, vascular, and inflammatory cells surrounded by immense quantities of extracellular matrix, far exceeding that found in most other tumor types. Remarkably, little is known about the composition and physiology of the PDAC tumor microenvironment, in particular, the role of stroma in tumor initiation and progression. This review attempts to define key challenges, opportunities and state-of-knowledge relating to the PDAC microenvironment research with an emphasis on how inflammatory processes and key cancer pathways may shape the ontogeny of the tumor stroma. Such knowledge may be used to understand the evolution and biology of this lethal cancer and may convert these insights into new points of therapeutic intervention.     

Prostate tumor-stroma interaction: molecular mechanisms and opportunities for therapeutic targeting

Maintenance of cell and tissue homeostasis is dependent upon the dynamic balance of cell proliferation, differentiation, and apoptosis through interactions between cells and their microenvironment. The unique prostatic cellular phenotypes are induced and maintained by interaction between epithelium and adjacent stroma through intimate intercellular signaling pathways. In this article, we summarize current advances in the tumor-stroma interaction and its biologic and therapeutic implications. We specifically emphasize current studies of the possible factors driving the "vicious cycle" between stroma and emerging prostate tumor epithelial cells that may be responsible for carcinogenesis and metastasis to bone. Stroma responds both genotypically and phenotypically to tumor epithelium upon co-culture under 3-D conditions. Likewise, the emerging carcinoma responds to stromal signals that drive progression to malignancy. A vicious cycle mediated by soluble and insoluble molecules secreted by tumor cells and stroma appear be the critical factors supporting and sustaining tumor colonization in bone. Co-targeting tumor and stroma with therapeutic agents has yielded promising results both in pre-clinical models of prostate cancer and bony metastasis and in clinical trials of patients treated with a dual tumor and stroma targeting strategies. In conclusion, understanding and targeting the interaction of the tumor and its stromal microenvironmant may improve the prognosis, reduce the suffering and increase the survival of patients with advanced cancer metastasis.     

Cadmium induces p53-dependent apoptosis in human prostate epithelial cells

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.     

The p53 tumor suppressor network in cancer and the therapeutic modulation of cell death

The molecular subversion of cell death is acknowledged as a principal contributor to the development and progression of cancer. The p53 tumor suppressor protein is among the most commonly altered proteins in human cancer. The p53 protein mediates critical functions within cells including the response to genotoxic stress, differentiation, senescence, and cell death. Loss of p53 function can result in enhanced rates of cell proliferation, resistance to cell death stimuli, genomic instability, and metastasis. The community of cancer scientists is now in possession of a vast repository of information regarding the frequency, specific mechanisms, and clinical context of cell death deregulation in cancer. This information has enabled the design of therapeutic agents to target proteins, including p53. The feasibility and impact of targeting cell death signaling proteins has been established in preclinical models of human cancer. The appropriate application of these targeted agents is now being established in clinical trials.     

Constitutively active DNA damage checkpoint pathways as the driving force for the high frequency of p53 mutations in human cancer

If the major function of the p53 protein is to function as a DNA damage checkpoint protein, then it is reasonable to hypothesize that its inactivation in human cancer must be related to its DNA damage checkpoint function. This hypothesis further implies that in tumor cells one or more of the DNA damage checkpoint pathways has been activated. Otherwise, p53 would not be active and there would be no selective pressure for TP53 mutations. I make the argument that tumorigenic transformation is intrinsically associated with formation of DNA DSBs in every cell cycle leading to activation of DNA damage checkpoint pathways. In turn, activation of the DNA DSB checkpoint provides the selective pressure for the high frequency of p53 inactivation in human cancer.     

High Lysyl Oxidase (LOX) in the Non-Malignant Prostate Epithelium Predicts a Poor Outcome in Prostate Cancer Patient Managed by Watchful Waiting

Lysyl oxidase (LOX) has been shown to both promote and suppress tumor progression, but its role in prostate cancer is largely unknown. LOX immunoreactivity was scored in prostate tumor epithelium, tumor stroma and in the tumor-adjacent non-malignant prostate epithelium and stroma. LOX scores in tumor and non-malignant prostate tissues were then examined for possible associations with clinical characteristics and survival in a historical cohort of men that were diagnosed with prostate cancer at transurethral resection and followed by watchful waiting. Men with a low LOX score in the non-malignant prostate epithelium had significantly longer cancer specific survival than men with a high score. Furthermore, LOX score in non-malignant prostate epithelium remained prognostic in a multivariable analysis including Gleason score. LOX score in prostate tumor epithelium positively correlated to Gleason score and metastases but was not associated with cancer survival. LOX score in tumor and non-malignant prostate stroma appeared unrelated to these tumor characteristics. In radical prostatectomy specimens, LOX immune-staining corresponded to LOX in-situ hybridization and LOX mRNA levels were found to be similar between tumor and adjacent non-malignant areas, but significantly increased in bone metastases samples. LOX levels both in tumors and in the surrounding tumor-bearing organ are apparently related to prostate cancer aggressiveness.