Joyce E. Rundhaug

Matrix metalloproteinases and angiogenesis.

Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissue. MMPs participate in this remodeling of basement membranes and ECM. However, it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM‐bound angiogenic growth factors, by exposing cryptic proangiogenic integrin binding sites in the ECM, by generating promigratory ECM component fragments, and by cleaving endothelial cell‐cell adhesions. MMPs can also contribute negatively to angiogenesis through the generation of endogenous angiogenesis inhibitors by proteolytic cleavage of certain collagen chains and plasminogen and by modulating cell receptor signaling by cleaving off their ligand‐binding domains. A number of inhibitors of MMPs that show antiangiogenic activity are already in early stages of clinical trials, primarily to treat cancer and cancer‐associated angiogenesis. However, because of the multiple effects of MMPs on angiogenesis, careful testing of these MMP inhibitors is necessary to show that these compounds do not actually enhance angiogenesis.

Cyclo‐oxygenase‐2 Plays a Critical Role in UV‐induced Skin Carcinogenesis

Besides induction of DNA damage and p53 mutations, chronic exposure to UV irradiation leads to the constitutive up‐regulation of cyclo‐oxygenase‐2 (COX‐2) expression and to increased production of its primary product in skin, prostaglandin E2 (PGE2). COX‐2 has also been shown to be constitutively overexpressed in mouse, as well as human, UV‐induced skin cancers and premalignant lesions. UV exposure results in ligand‐independent activation of the epidermal growth factor receptor and subsequent activation of mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase/Akt pathways leading to transcriptional activation of the COX‐2 gene. Use of COX‐2‐specific inhibitors and genetic manipulation of COX‐2 expression have demonstrated that UV induction of COX‐2 in the skin contributes to the induction of epidermal hyperplasia, edema, inflammation, and counters the induction of apoptosis after UV exposure. Likewise, inhibition of COX‐2 activity or reduced expression in COX‐2 knockout mice resulted in significantly reduced UV‐induced tumorigenesis, while overexpression of COX‐2 in transgenic mice enhanced UV‐induced tumor development. A combination of signaling from the PGE2 EP1, EP2 and/or EP4 receptors mediates the effects of COX‐2 overexpression. These studies demonstrate the crucial role of COX‐2 in the development of UV‐related nonmelanoma skin cancers.

Cyclooxygenase-2 expression is critical for chronic UV-induced murine skin carcinogenesis.

While it has been established that both the constitutive and inducible forms of cyclooxygenase (COX‐1 and COX‐2, respectively) play important roles in chemical initiation‐promotion protocols with phorbol ester tumor promoters, the contribution of these two enzymes to ultraviolet (UV) light‐induced skin tumors has not been fully assessed. To better understand the contribution of COX‐1 and COX‐2 to UV carcinogenesis, we transferred the null allele for each isoform onto the SKH‐1 hairless strain of mouse. Due to low viability on this background with complete knockout of COX‐2, heterozygous mice were used in UV carcinogenesis experiments. While the lack of one allele of COX‐1 had no effect on tumor outcome, the lack of one allele of COX‐2 resulted in a 50–65% reduction in tumor multiplicity and a marked decrease in tumor size. Additionally, transgenic SKH‐1 mice that overexpress COX‐2 under the control of a keratin 14 promoter developed 70% more tumors than wild‐type SKH‐1 mice. The lack of one allele of either COX‐1 or COX‐2 reduced prostaglandin (PG) E2 levels in response to a single UV treatment. The proliferative response to UV was significantly reduced in COX‐2, but not COX‐1, heterozygous mice. UV‐induced apoptosis, however, was greater in COX‐2 heterozygous mice. Collectively, these results clearly establish the requirement for COX‐2 in the development of skin tumors.

Molecular Mechanisms of Mouse Skin Tumor Promotion

Multiple molecular mechanisms are involved in the promotion of skin carcinogenesis. Induction of sustained proliferation and epidermal hyperplasia by direct activation of mitotic signaling pathways or indirectly in response to chronic wounding and/or inflammation, or due to a block in terminal differentiation or resistance to apoptosis is necessary to allow clonal expansion of initiated cells with DNA mutations to form skin tumors. The mitotic pathways include activation of epidermal growth factor receptor and Ras/Raf/mitogen-activated protein kinase signaling. Chronic inflammation results in inflammatory cell secretion of growth factors and cytokines such as tumor necrosis factor-α and interleukins, as well as production of reactive oxygen species, all of which can stimulate proliferation. Persistent activation of these pathways leads to tumor promotion.

A role for cyclooxygenase-2 in ultraviolet light-induced skin carcinogenesis

Nonmelanoma skin cancer is the most prevalent cancer in the United States and its incidence is on the rise. These cancers generally arise on sun‐exposed areas of the body and the ultraviolet (UV) B spectrum of sunlight has been clearly identified as the major carcinogen responsible for skin cancer development. Besides inducing DNA damage directly, UV exposure of the skin induces the expression of the enzyme cyclooxygenase‐2 (COX‐2), which catalyzes the first step in the conversion of arachidonic acid to prostaglandins, the primary product in skin being prostaglandin E2 (PGE2). COX‐2 has been shown to be overexpressed in premalignant lesions as well as in nonmelanoma skin cancers in both humans and mice chronically exposed to UV. Through the use of COX‐2‐selective inhibitors and COX‐2 knockout mice, it has been shown that UV‐induced COX‐2 expression plays a major role in UV‐induced PGE2 production, inflammation, edema, keratinocyte proliferation, epidermal hyperplasia, and generation of a pro‐oxidant state leading to oxidative DNA damage. Chronic exposure to UV leads to chronic up‐regulation of COX‐2 expression and chronic inflammation along with the accumulation of DNA damage and mutations, all of which combine to induce malignant changes in epidermal keratinocytes and skin cancers. Both inhibition of COX‐2 activity and reduction in COX‐2 expression by genetic manipulations significantly reduce, while overexpression of COX‐2 in transgenic mice significantly increases UV‐induced skin carcinogenesis. Together these studies demonstrate that COX‐2 expression/activity is critical to the development of UV‐related nonmelanoma skin cancers.

Transcriptional regulation of estrogen receptor-alpha by p53 in human breast cancer cells.

Estrogen receptor alpha (ER) and p53 are critical prognostic indicators in breast cancer. Loss of functional p53 is correlated with poor prognosis, ER negativity, and resistance to antiestrogen treatment. Previously, we found that p53 genotype was correlated with ER expression and response to tamoxifen in mammary tumors arising in mouse mammary tumor virus-Wnt-1 transgenic mice. These results lead us to hypothesize that p53 may regulate ER expression. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated a 5-fold increase in p53 expression. ER expression was also increased 4-fold over a 24-h time frame. In cells treated with small interfering RNA (siRNA) targeting p53, expression of both p53 and ER was significantly reduced (>60%) by 24 h. Induction of ER by DNA-damaging agents was p53 dependent as either ionizing radiation or doxorubicin failed to up-regulate ER after treatment with p53-targeting siRNA. To further investigate whether p53 directly regulates transcription of the ER gene promoter, MCF-7 cells were transiently transfected with a wild-type (WT) p53 expression vector along with a luciferase reporter containing the proximal promoter of ER. In cells transfected with WT p53, transcription from the ER promoter was increased 8-fold. Chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun, and Sp1 and that this multifactor complex was formed in a p53-dependent manner. These data show that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer.

The role of the EP receptors for prostaglandin E2 in skin and skin cancer

One of the most common features of exposure of skin to ultraviolet (UV) light is the induction of inflammation, a contributor to tumorigenesis, which is characterized by the synthesis of cytokines, growth factors and arachidonic acid metabolites, including the prostaglandins (PGs). Studies on the role of the PGs in non-melanoma skin cancer (NMSC) have shown that the cyclooxygenase-2 (COX-2) isoform of the cyclooxygenases is responsible for the majority of the pathological effects of PGE2. In mouse skin models, COX-2 deficiency significantly protects against chemical carcinogen- or UV-induced NMSC while overexpression confers endogenous tumor promoting activity. Current studies are focused on identifying which of the G protein-coupled EP receptors mediate the tumor promotion/progression activities of PGE2 and the signaling pathways involved. As reviewed here, the EP1, EP2, and EP4 receptors, but not the EP3 receptor, contribute to NMSC development, albeit through different signaling pathways and with somewhat different outcomes. The signaling pathways activated by the specific EP receptors are context specific and likely depend on the level of PGE2 synthesis, the differential levels of expression of the different EP receptors, as well as the levels of expression of other interacting receptors. Understanding the role and mechanisms of action of the EP receptors potentially offers new targets for the prevention or therapy of NMSCs.

Multiple Signaling Pathways Are Responsible for Prostaglandin E2–Induced Murine Keratinocyte Proliferation

Although prostaglandin E2 (PGE2) has been shown by pharmacologic and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor growth is not well understood. In this study, we investigated the mechanisms by which PGE2 stimulates murine keratinocyte proliferation using in vitro and in vivo models. In primary mouse keratinocyte cultures, PGE2 activated the epidermal growth factor receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP2), nor by a protein kinase A inhibitor (H-89). However, PGE2-stimulated extracellularly regulated kinase 1/2 (ERK1/2) activation was completely blocked by EGFR, ERK1/2, and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors. In addition, these inhibitors attenuated the PGE2-induced proliferation, nuclear factor-κB, activator protein-1 (AP-1), and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF in primary mouse keratinocytes. Similarly, in vivo, we found that WT mice treated with PGE2 and untreated cyclooxygenase-2–overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, nuclear factor-κB, AP-1, and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between cyclooxygenase-2 overexpression and EGFR-, ERK-, PI3K-, cAMP-mediated cell proliferation, and the tumor-promoting activity of PGE2 in mouse skin. (Mol Cancer Res 2008;6(6):1003–16)

Paracrine Overexpression of Insulin-Like Growth Factor-1 Enhances Mammary Tumorigenesis in Vivo

Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.

The effect of cyclooxygenase-2 overexpression on skin carcinogenesis is context dependent.

The up‐regulation of the inducible form of cyclooxygenase (COX‐2), a central enzyme in the prostaglandin (PG) biosynthetic pathway, occurs in many epithelial tumors and has been associated with tumor cell proliferation and angiogenesis. To better understand the role of COX‐2 in skin tumor development, we generated transgenic mice that overexpress COX‐2 under the control of the keratin 14 promoter. We previously reported (Cancer Res. 62: 2516, 2002) that these mice, referred to as keratin 14 (K14).COX2 mice, were unexpectedly very resistant to 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) tumor promotion. The current studies were undertaken to determine the mechanism of this resistance and determine if it was restricted to TPA promotion. Transgenic and wild‐type mice were subjected to a complete carcinogenesis protocol using 7,12‐dimethylbenz[a]anthracene (DMBA) only, as well as a two‐stage protocol using DMBA plus an unrelated tumor promoter, anthralin. In addition, the responses of transgenic and wild‐type mice to TPA in terms of induction of proliferation and various down‐stream mediators were examined. The TPA resistance phenotype correlated with a reduced ability to induce ornithine decarboxylase, interleukin‐1α, and tumor necrosis factor‐α and a reduced proliferation response. This resistance phenotype appears to be restricted to phorbol ester promotion because K14.COX2 mice developed six times more tumors than wild‐type mice when anthralin was used as the tumor promoter. Additionally, K14.COX2 mice treated only with DMBA developed approximately 3.5 times more tumors than wild‐type mice, suggesting that PGs have intrinsic tumor promoting activity. We conclude that the role of PGs in skin tumorigenesis is context dependent.