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Activated p53 suppresses the histone methyltransferase EZH2 gene

Replicative senescence is an irreversible cell cycle arrest that limits the proliferation of damaged cells and may be an important tumor suppression mechanism in vivo. This process is regulated at critical steps by the tumor suppressor p53. To identify genes that may regulate the senescence process, we performed cDNA microarray analysis of gene expression in senescent, young proliferating, and hTERT-immortalized primary human fibroblasts. The histone methyltransferase (HMTase), EZH2, was specifically downregulated in senescent cells. Activated p53 suppressed EZH2 gene expression through repression of the EZH2 gene promoter. This activity of p53 requires intact p53 transactivation and DNA binding domains. Furthermore, the repression of EZH2 promoter by p53 is dependent on p53 transcriptional target p21Waf1 inactivating RB/E2F pathways. In addition, the knockdown of EZH2 expression retards cell proliferation and induces G2/M arrest. We suggest that the p53-dependent suppression of EZH2 expression is a novel pathway that contributes to p53-mediated G2/M arrest. EZH2 associated complex possesses HMTase activity and is involved in epigenetic regulation. Activated p53 suppresses EZH2 expression, suggesting a further role for p53 in epigenetic regulation and in the maintenance of genetic stability. Suppression of EZH2 expression in tumors by p53 may lead to novel approaches to control cancer progression.

From sentinel cells to inflammatory culprits: cancer-associated fibroblasts in tumour-related inflammation.

Inflammation is now established as a hallmark of cancer. Cancer‐associated fibroblasts (CAFs) have been established as a key component of the crosstalk between tumour cells and their microenvironment. Central to the role of CAFs in facilitating tumour growth, invasion, and metastasis is their ability to orchestrate tumour‐related inflammation. CAFs and their soluble mediators provide multiple complex regulatory signals that modulate the trafficking, differentiation status, and function of inflammatory cells in the tumour microenvironment. This review focuses on pathways by which CAFs mediate tumour‐promoting inflammation and modify the components of the inflammatory microenvironment that facilitate tumour initiation, progression, and metastasis.Copyright

Structural and functional involvement of p53 in BER in vitro and in vivo.

p53 is involved in several DNA repair pathways. Some of these require the specific transactivation of p53-dependent genes and others involve direct interactions between the p53 protein and DNA repair associated proteins. Previously, we have shown that p53 acts directly in Base Excision Repair (BER) when assayed under in vitro conditions. Our present data indicate that this involvement is independent of the transcriptional activity of the p53 molecule. We found that under both in vitro and in vivo conditions, a p53 transactivation-deficient molecule, p53-22-23 was more efficient in BER activity than was wild type p53. However, mutations in the core domain or C-terminal alterations strongly reduced p53-mediated BER activity. These results are consistent with the hypothesis that the involvement of p53 in BER activity, a housekeeping DNA repair pathway, is a prompt and immediate one that does not involve the activation of p53 transactivation-dependent mechanisms, but rather concerns with the p53 protein itself. In an endogenous DNA damage status p53 is active in BER pathways as a protein and not as a transcription factor.

Cancer associated fibroblasts express pro-inflammatory factors in human breast and ovarian tumors.

Inflammation has been established in recent years as a hallmark of cancer. Cancer Associated Fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation and invasion. We previously demonstrated that CAFs also mediate tumor-enhancing inflammation in a mouse model of skin carcinoma. Breast and ovarian carcinomas are amongst the leading causes of cancer-related mortality in women and cancer-related inflammation is linked with both these tumor types. However, the role of CAFs in mediating inflammation in these malignancies remains obscure. Here we show that CAFs in human breast and ovarian tumors express high levels of the pro-inflammatory factors IL-6, COX-2 and CXCL1, previously identified to be part of a CAF pro-inflammatory gene signature. Moreover, we show that both pro-inflammatory signaling by CAFs and leukocyte infiltration of tumors are enhanced in invasive ductal carcinoma as compared with ductal carcinoma in situ. The pro-inflammatory genes expressed by CAFs are known NF-κB targets and we show that NF-κB is up-regulated in breast and ovarian CAFs. Our data imply that CAFs mediate tumor-promoting inflammation in human breast and ovarian tumors and thus may be an attractive target for stromal-directed therapeutics.

Transcriptional Programs following Genetic Alterations in p53, INK4A, and H-Ras Genes along Defined Stages of Malignant Transformation

The difficulty to dissect a complex phenotype of established malignant cells to several critical transcriptional programs greatly impedes our understanding of the malignant transformation. The genetic elements required to transform some primary human cells to a tumorigenic state were described in several recent studies. We took the advantage of the global genomic profiling approach and tried to go one step further in the dissection of the transformation network. We sought to identify the genetic signatures and key target genes, which underlie the genetic alterations in p53, Ras, INK4A locus, and telomerase, introduced in a stepwise manner into primary human fibroblasts. Here, we show that these are the minimally required genetic alterations for sarcomagenesis in vivo. A genome-wide expression profiling identified distinct genetic signatures corresponding to the genetic alterations listed above. Most importantly, unique transformation hallmarks, such as differentiation block, aberrant mitotic progression, increased angiogenesis, and invasiveness, were identified and coupled with genetic signatures assigned for the genetic alterations in the p53, INK4A locus, and H-Ras, respectively. Furthermore, a transcriptional program that defines the cellular response to p53 inactivation was an excellent predictor of metastasis development and bad prognosis in breast cancer patients. Deciphering these transformation fingerprints, which are affected by the most common oncogenic mutations, provides considerable insight into regulatory circuits controlling malignant transformation and will hopefully open new avenues for rational therapeutic decisions.

Melanoma miRNA trafficking controls tumour primary niche formation

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

Leukocytes as paracrine regulators of metastasis and determinants of organ-specific colonization

It is now well recognized that tumor cell–host interactions regulate all aspects of cancer development. Amongst the various host response programs that facilitate primary cancer development, an emerging body of literature points to a critical role for leukocytes and their soluble mediators as regulating discrete events during primary tumor development and metastasis. This review focuses on the multiple aspects of leukocytes and their effector molecules as regulators of the metastatic process.

Tumor-Derived Osteopontin Reprograms Normal Mammary Fibroblasts to Promote Inflammation and Tumor Growth in Breast Cancer

Breast tumors are characterized by an extensive desmoplastic stroma, abundantly populated by fibroblasts. Cancer-associated fibroblasts (CAF) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. CAF also orchestrate tumor-promoting inflammation in multiple tumor types, including breast cancer. However, the mechanisms through which normal tissue fibroblasts are reprogrammed to tumor-promoting CAFs are mainly obscure. Here, we show that mammary fibroblasts can be educated by breast cancer cells to become activated to a proinflammatory state that supports malignant progression. Proteomic analysis of breast cancer cell-secreted factors identified the secreted proinflammatory mediator osteopontin, which has been implicated in inflammation, tumor progression, and metastasis. Osteopontin was highly secreted by mouse and human breast cancer cells, and tumor cell-secreted osteopontin activated a CAF phenotypes in normal mammary fibroblasts in vitro and in vivo. Osteopontin was sufficient to induce fibroblast reprogramming and neutralizing antibodies against osteopontin-blocked fibroblast activation induced by tumor cells. The ability of secreted osteopontin to activate mammary fibroblasts relied upon its known receptors CD44 and αVβ3 integrin. Strikingly, osteopontin silencing in tumor cells in vivo attenuated stromal activation and inhibited tumor growth. Our findings establish a critical functional role for paracrine signaling by tumor-derived osteopontin in reprograming normal fibroblasts into tumor-promoting CAFs.

Astrocytes facilitate melanoma brain metastasis via secretion of IL-23.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain‐metastasizing melanoma cells to express pro‐inflammatory factors, including the cytokine IL‐23, which was highly expressed by metastases‐associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up‐regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain‐metastasizing melanoma cells. IL‐23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL‐23 could block this enhanced migration, implying a functional role for astrocyte‐derived IL‐23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL‐23‐induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro‐inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells. Copyright

An inflammatory vicious cycle: Fibroblasts and immune cell recruitment in cancer

Cancer-associated fibroblasts (CAFs) have been established as a key component of the crosstalk between tumor cells and their microenvironment. The ability of CAFs to orchestrate tumor-promoting inflammation is central to their role in facilitating tumor growth, invasion, and metastasis. Here we review pathways by which CAFs and their soluble mediators provide multiple complex signals that modulate the recruitment, functional activation status, and retention of immune cells in the tumor microenvironment.