Retinoblastoma protein and MyoD function together to effect the repression of Fra-1 and in turn cyclin D1 during terminal cell cycle arrest associated with myogenesis.

The acquisition of skeletal muscle-specific function and terminal cell cycle arrest represent two important features of the myogenic differentiation program. These cellular processes are distinct and can be separated genetically. The lineage-specific transcription factor MyoD and the retinoblastoma protein pRb participate in both of these cellular events. Whether and how MyoD and pRb work together to effect terminal cell cycle arrest is uncertain. To address this question, we focused on cyclin D1, whose stable repression is required for terminal cell cycle arrest and execution of myogenesis. MyoD and pRb are both required for the repression of cyclin D1; their actions, however, were found not to be direct. Rather, they operate to regulate the immediate early gene Fra-1, a critical player in mitogen-dependent induction of cyclin D1. Two conserved MyoD-binding sites were identified in an intronic enhancer of Fra-1 and shown to be required for the stable repression of Fra-1 and, in turn, cyclin D1. Localization of MyoD alone to the intronic enhancer of Fra-1 in the absence of pRb was not sufficient to elicit a block to Fra-1 induction; pRb was also recruited to the intronic enhancer in a MyoD-dependent manner. These observations suggest that MyoD and pRb work together cooperatively at the level of the intronic enhancer of Fra-1 during terminal cell cycle arrest. This work reveals a previously unappreciated link between a lineage-specific transcription factor, a tumor suppressor, and a proto-oncogene in the control of an important facet of myogenic differentiation.

Cyclin D1 and C/EBPß LAP1 operate in a common pathway to promote mammary epithelial cell differentiation.

Both cyclin D1 and the transcription factor C/EBPß are required for mammary epithelial cell differentiation; however, the pathway in which they operate is uncertain. Previous analyses of the patterns of gene expression in human tumors suggested a connection between cyclin D1 overexpression and C/EBPß, but whether this represents a cancer-specific gain of function for cyclin D1 is unknown. C/EBPß is an intronless gene encoding three protein isoforms--LAP1, LAP2, and LIP. Here, we provide evidence that cyclin D1 engages C/EBPß in an isoform-specific manner. Cyclin D1 binds to LAP1, an event that activates the transcriptional function of LAP1 by relieving its autoinhibited state effected by intramolecular interactions. Reexpression of LAP1 but not LAP2 or LIP restores the ability of C/EBPß-deficient mammary epithelial cells to differentiate and does so in a manner dependent on cyclin D1. And cyclin D1-mediated activation of LAP1 participates in mammary epithelial cell differentiation. Our findings indicate that cyclin D1 and C/EBPß LAP1 operate in a common pathway to promote mammary epithelial cell differentiation.

CCL18 from tumor-associated macrophages promotes breast cancer metastasis via PITPNM3.

Tumor-associated macrophages (TAMs) can influence cancer progression and metastasis, but the mechanism remains unclear. Here, we show that breast TAMs abundantly produce CCL18, and its expression in blood or cancer stroma is associated with metastasis and reduced patient survival. CCL18 released by breast TAMs promotes the invasiveness of cancer cells by triggering integrin clustering and enhancing their adherence to extracellular matrix. Furthermore, we identify PITPNM3 as a functional receptor for CCL18 that mediates CCL18 effect and activates intracellular calcium signaling. CCL18 promotes the invasion and metastasis of breast cancer xenografts, whereas suppressing PITPNM3 abrogates these effects. These findings indicate that CCL18 derived from TAMs plays a critical role in promoting breast cancer metastasis via its receptor, PITPNM3.

Wild-type NRas and KRas perform distinct functions during transformation.

The ras proto-oncogenes, of which there are four isoforms, are molecular switches that function in signal transduction pathways to control cell differentiation, proliferation, and survival. How the Ras isoforms orchestrate cellular processes that affect behavior is poorly understood. Further, why cells express two or more Ras isoforms is unknown. Here, using a genetically defined system, we show that the presence of both wild-type KRas and NRas isoforms is required for transformation because they perform distinct nonoverlapping functions: wild-type NRas regulates adhesion, and KRas coordinates motility. Remarkably, we find that Ras isoforms achieve functional specificity by engaging different signaling pathways to affect the same cellular processes, thereby coordinating cellular outcome. Although we find that signaling from both isoforms intersects in actin and microtubule cytoskeletons, our results suggest that KRas signals through Akt and Cdc42 while NRas signals through Raf and RhoA. Our analyses suggest a previously unappreciated convergence of different Ras isoforms on the dynamics of the processes involved in transformation.

Genetic interaction between Rb and N-ras: differentiation control and metastasis.

The retinoblastoma tumor suppressor gene, Rb, and the ras proto-oncogenes regulate various cellular processes, including differentiation and proliferation. Rb and ras genetically interact to positively influence differentiation in the mouse. This genetic interaction between Rb and ras also affects tumor development, either positively or negatively depending on cell type. Loss of one or two N-ras alleles allows medullary thyroid (C cell) adenomas occurring in Rb heterozygous mice to progress to metastatic carcinomas, an event associated with C cells displaying a less-differentiated phenotype. Here, we discuss the genetic interaction between Rb and ras and the development of a mouse model of medullary thyroid carcinoma.

Nras loss induces metastatic conversion of Rb1-deficient neuroendocrine thyroid tumor.

Mutations in the gene encoding the retinoblastoma tumor suppressor predispose humans and mice to tumor development. Here we have assessed the effect of Nras loss on tumor development in Rb1 heterozygous mice. Loss of one or two Nras alleles is shown to significantly reduce the severity of pituitary tumors arising in Rb1(+/-) animals by enhancing their differentiation. By contrast, C-cell thyroid adenomas occurring in Rb1(+/-) mice progress to metastatic medullary carcinomas after loss of Nras. In Rb1(+/-)Nras(+/-) animals, distant medullary thyroid carcinoma metastases are associated with loss of the remaining wild-type Nras allele. Loss of Nras in Rb1-deficient C cells results in elevated Ras homolog family A (RhoA) activity, and this is causally linked to the invasiveness and metastatic behavior of these cells. These findings suggest that the loss of the proto-oncogene Nras in certain cellular contexts can promote malignant tumor progression.

Genetic interaction between Rb and K-ras in the control of differentiation and tumor suppression.

Although the retinoblastoma protein (pRb) has been implicated in the processes of cellular differentiation, there is no compelling genetic or in vivo evidence that such activities contribute to pRb-mediated tumor suppression. Motivated by cell culture studies suggesting that Ras is a downstream effector of pRb in the control of differentiation, we have examined the tumor and developmental phenotypes of Rb and K-ras double-knockout mice. We find that heterozygosity for K-ras (i) rescued a unique subset of developmental defects that characterize Rb-deficient embryos by affecting differentiation but not proliferation and (ii) significantly enhanced the degree of differentiation of pituitary adenocarcinomas arising in Rb heterozygotes, leading to their prolonged survival. These observations suggest that Rb and K-ras function together in vivo, in the contexts of both embryonic and tumor development, and that the ability to affect differentiation is a major facet of the tumor suppressor function of pRb.

The activities of cyclin D1 that drive tumorigenesis.

The proto-oncogene cyclin D1 has been implicated in the genesis of a large proportion of human tumors from diverse histological origins. It has long been assumed that the action of cyclin D1, as an activator of cdk4 and cdk6 and leading to progression through the G1 phase of the cell cycle, underlies its pathological activity. But, more recently, analyses of the patterns of gene expression in human cancer have revealed a previously unappreciated mechanism of action for cyclin D1, suggesting that both cdk-dependent and cdk-independent activities might contribute to tumorigenesis. The development of therapeutics designed to target the aberrant activity of cyclin D1 in human cancers will rely upon an intimate molecular understanding of these distinct mechanisms of actions and their relative importance. Here, we describe the known functions of the cyclin D1 oncogene and delineate the evidence that cdk-independent actions are important for cyclin D1-mediated oncogenesis.

Cyclin D1 and molecular chaperones: implications for tumorigenesis.

We recently investigated the mechanisms of cyclin D1 action in human cancer using global analyses of gene expression. With an experimentally-determined expression signature for cyclin D1 overexpression, gene expression data from human tumors, and a novel data-mining method, we were able to reveal a previously unappreciated and apparently predominant functional interdependency between cyclin D1 and C/EBPbeta. Many of the genes we found to be affected by cyclin D1 overexpression are recognized as molecular chaperones or their regulators. Might this provide insights to the role of the cyclin D1-C/EBPbeta axis in carcinogenesis?

A mechanism of cyclin D1 action encoded in the patterns of gene expression in human cancer.

Here we describe how patterns of gene expression in human tumors have been deconvoluted to reveal a mechanism of action for the cyclin D1 oncogene. Computational analysis of the expression patterns of thousands of genes across hundreds of tumor specimens suggested that a transcription factor, C/EBPbeta/Nf-Il6, participates in the consequences of cyclin D1 overexpression. Functional analyses confirmed the involvement of C/EBPbeta in the regulation of genes affected by cyclin D1 and established this protein as an indispensable effector of a potentially important facet of cyclin D1 biology. This work demonstrates that tumor gene expression databases can be used to study the function of a human oncogene in situ.

Rb and N-ras function together to control differentiation in the mouse.

The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote differentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine embryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the developmental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb(-/-); N-ras(-/-) skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb(-/-); N-ras(-/-) muscle shows a restoration in the expression of the late muscle-specific gene MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb(-/-); N-ras(-/-), compared to Rb(-/-) myoblasts in culture. The improved differentiation of skeletal muscle in Rb(-/-); N-ras(-/-) embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable.