The Role of Endothelin in Cancer
With a Focus on Prostate Cancer and Breast Cancer

Francesca Leo
Megan Maragakes
Aki Niihara
Taylor Pecora-Saipe

Abstract:

Purpose: This paper was written to make the reader more aware of the role endothelin plays in cancer, specifically prostate cancer and breast cancer. Endothelin-1 functions in the following ways: as a vasomotor regulator, cell proliferator, hormone producer, tissue differentiator and developer. In prostate and breast cancer, endothelin is overproduced which leads to metastasization. The history of endothelin, its role in prostate and breast cancer, and new research methods to regulate endothelin production and eliminate carcinoma in the affected areas are reviewed in this paper.
Materials and Methods: A review was conducted on published data in relationship to endothelin and its role in prostate and breast cancer, including research in treatment.

Results: It has been noted that an overproduction of endothelin is a key factor in the metastasis of prostate and breast cancer. Studies have been conducted on drugs that target the endothelin axis and have been recorded as playing a major role in the digression of these diseases.
Conclusions: Although methods to regulate the production of endothelin have been underway and seemingly successful, more clinical research and lab work need to be completed before the problem can be considered obsolete.

Key words: endothelin, endothelin receptors, breast neoplasm, prostate neoplasm, metastasis, angiogenesis, apoptosis, atrasentan, clinical trials, bone and bones.

Introduction:

In 1988, a potent vasoconstrictor called endothelin, which is released from endothelial cells, was isolated.1 The most common type of endothelin is ET-1; there is also an ET-2 and an ET-3. ET-1 is found primarily in endothelial cells, while ET-2 is present in the kidney and intestines, and ET-3 appears mainly in the brain. The ETs and their receptors, ETa and ETb, make up what is referred to as the endothelin axis.

      Endothelin (ET-1) is made up of twenty-one amino acid residues which are produced in the cells and tissues of the body(Figure 1).2 The main functions of endothelin are: the regulation of vasomotor activity, cell proliferation, the production of hormones, tissue differentiation and development(Figure 2).2 Endothelin is produced by endothelium, a thin layer of flat epithelial cells, which releases an endothelin precursor made of thirty-nine amino acids. This precursor is then cleaved into ET-1, as a result of an endothelin converting enzyme (ECE), which is found on the membrane of endothelial cells. Then ET-1 binds to ETa receptors, which are found on contiguous vascular smooth muscle cells. This reaction triggers calcium mobilization and the contraction of smooth muscle, or vasoconstriction. However, ET-1 can also bind to ETb receptors, which are also situated in the vascular endothelium. This reaction results in the release of nitric oxide, a vasodilator, by the endothelium; therefore, nitric oxide inhibits vasoconstriction.3,4

     It has been discovered recently that the endothelin axis plays a role in mitogenesis, apoptosis inhibition, invasiveness, angiogenesis, and bone remodeling. Proliferation of ET-1 contributes to angiogenesis, the formation of new blood vessels. The stages of angiogenesis include: rapid increase of endothelial-cell production, migration and invasion of blood vessels, and tube formation of the blood vessels. These stages are facilitated by endothelin. As a result of growth factors, namely endothelin, secreted from endothelial cells, angiogenesis is increased in malignant tumors. The vascular endothelial growth factor (VEGF) proliferates by the formation of the ET-1/ETA complex.2

(Figure 3)

      Current research has determined that endothelin plays a major role in the growth and progression of malignant tumors. An overabundance of ETa in the body aids in the formation of tumors, while a depression in ETb increases pain in the bones of individuals affected by cancer. ET receptor activation promotes malignancies by a variety of mechanisms including cell proliferation, inhibition of cell death, and bone metastases by activating osteoblasts. Without a vital mechanism such as apoptosis, for example, cancer cells are unable to die and thus continue to grow and spread.2 Two main focuses of the relationship between endothelin and cancer have been on prostate and breast neoplasms. The overproduction of ET-1 contributes to the creation of cancer within either the breast in females or the prostate in males.

      Urologist Joel Nelson, M.D., was the first to discover the relationship between endothelin and prostate cancer.5 Semen is a fluid that is partly secreted by the prostate. Nelson found that the concentration of endothelin is about 500 times greater in the semen of men with prostate cancer than in men unaffected by prostate cancer. Nelson has also tied endothelin to the bone damage seen in some prostate cancer patients. This is when the bone becomes unnaturally hard and thick and causes severe pain for the patient. Nelson’s belief that this pain could be eliminated by the use of an endothelin-receptor antagonist has not been tested until recently; presently, clinical trials are being performed on an endothelin-receptor antagonist, or an endothelin-blocking agent. Two antagonists that have yielded positive results thus far are atrasentan (ABT-627) and Xinlay. It is believed that these bioavailable endothelin-blocking agents will inhibit the overproduction of ET-1, therefore avoiding the formation of malignant tumors. It may also prevent the spread of cancer to the bones.6

Endothelin in Prostate Cancer:

     Endothelin-1 (ET-1) is a common substance in prostatic epithelium. In unaffected men, the mean plasma ET-1 concentration is 5.1 pg/mL, whereas in men with prostate cancer, the mean concentration is 13.2 pg/mL.6 It has also been determined that the enzyme which is supposed to degrade ET-1, neutral endopeptidase 24.11, is less prominent in prostate cancer.7 Therefore, there is an overproduction of ET-1 in men with prostate cancer.

      ET_A and ET_B are G protein-coupled receptors that intercede the actions of ET-1. ET_B is the predominant endothelin receptor in healthy prostatic epithelium and is also responsible for the regulation of ET-1 production. In prostate cancer, however, there is a significant reduction in expression of ETB. It is thought that the reduction in the expression of ETB is due to the rapid addition of methyl groups (hypermethylation) to the ETB receptor gene, EDNRB. In the promoter region of the EDNRB gene, there is an abundance of dinucleotides cytosine followed by guanine; this combination is called a CpG island. When a methyl group is added to the CpG island, gene transcription is inactivated, thus silencing ETB expression. Reduction in ETB initiates the production of ETA. The ET-1/ ETA axis consequently causes the concentration of ET-1 to increase without limitation, leading to prostate cancer progression.

     ET-1 plays several roles in prostate cancer development. As a weak inducer of mitosis, it minimally encourages the production of prostate cancer cells. By stimulating alternate growth factors, it promotes proliferation of prostate cancer cells. Additionally, ET-1 is responsible for the inhibition of prostate cancer apoptosis, or programmed cell death.

     One of the most noteworthy characteristics of prostate cancer is the formation of bone metastases, or the spread of tumorgenic cells.8 It has been found that in the presence of increased concentrations of ET-1, the mitogenic activity of osteoblasts increases. The increased concentrations of ET-1 also contribute to the inhibition of osteoclast function, or the reabsorption of bone. As a result of this imbalance in osteoblast–osteoclast activity, bone is produced in tissues surrounding prostate cancer cells, causing severe pain for the infected individual.9

      In numerous experiments conducted on animals (species unspecified in collected materials), ET-1 was observed as playing a prominent role in the osteoblastic activity of bone, which is problematic. As a solution to the osteoblastic activity, experiments have concluded that ETA antagonists blocked the osteoblastic activity of bone, which is beneficial to the prostate cancer patient. In an experiment performed on animals (species unspecified in collected materials), ETA receptor antagonists prevented ET-1-induced pain reactions. As a result of these and other observations, it has been hypothesized that if ET-1 is a factor in the development of prostate cancer, then concentration on the ETA receptor would be beneficial in fighting prostate cancer.10

Endothelin in Breast Cancer

     Breast epithelial cells regularly produce and secrete ET-1 in small quantities. Similar to the excessively high concentrations of endothelin in prostate cancer cells, endothelin and its receptors are overly expressed in breast cancer. Different functions that involve the endothelin axis in breast cancer are transformation, differentiation, and growth processes in the human breast. In addition, ET-1 is involved in tumor progression and stimulates tumor cell growth, invasion, angiogenesis, and neovascularization. ET-1 also triggers mitosis of several cell types including human breast fibroblasts and breast cancer cells.11
     Alanen et al. compared the distribution of ET-1, ET-3, and mRNAs for ET-1, ET-3, ETA, and ETB in normal breast tissue and breast carcinomas. The results of the study showed significantly higher concentrations of ET-1 and ET-3 mRNAs present in malignant breast tumors compared to normal breast tissue; in all of the normal breast tissue, staining for both ET-1 and ET-3 were categorized as grade I (less than 25% cytoplasmic positivity of tumor cells), while in the cancerous tissues, 42% of the cases showed grade II (25-75% cytoplasmic positivity) staining ET-1 and 58% of the cases showed grade III (75-100% cytoplasmic positivity) staining for ET-3. Southern hybridizations, an evaluative procedure used in this experiment, also demonstrated a greater concentration of ETB receptor mRNAs in the carcinoma tissue samples than in the normal tissue samples.11

      Grimshaw et al. found that endothelin stimulates breast tumor cells to adopt chemotactic, a tendency to move in response to chemical attractions, and invasive properties. All of the breast tumor cell lines (MCF-7, Murine HTH-K, MDAMB468, SKBR3, and BT20) moved most significantly toward the 100 ng/ml of ET-2 than toward either the ET-1 or ET-3. By adding endothelins to the lower wells of an invasion chamber during a 96 hour in vitro invasion trial, it was found that ET-1 and ET-2 stimulated tumor cells to become aggressively invasive. Finally, when endothelin was added to a coculture of macrophages and tumor cells, the tumor cell lines became notably invasive. Under normal circumstances, macrophages are known to inhibit the effects of foreign substances. However, when ET-1 and ET-2 promote invasive behavior in tumor cells, macrophages were found to produce a substance that encourages tumor cell invasion. Clearly, endothelin and its receptors are pivotal in the progression of breast cancer and the invasion of breast cancer tumor cells.12

      The increase in endothelin is due to various stimuli including hypoxia, growth factors, and cytokines13,14. TGF-?, IL-6, cortisol, bombesin, and prolactin are also considered factors responsible for endothelin overproduction in breast cancer cells.12 There is evidence for participation of the ET axis in blood perfusion of breast tumors because in a rodent model blood flow to the tumor tissue increased significantly in response to ET-1 mediated through ETB receptor.15 Increased ET-1, ETA and ETB receptor expression is particularly present in cancerous breast tumors that have a high vascularity, or blood supply, and could be involved in the regulation of angiogenesis.15 A specific regulator of angiogenesis has not yet been identified. However, studies showed that in the presence of angiogenic factors, such as VEGF, bFGF and PD-ECGF, the breast cancer is more malignant.16

      The concentrations of ET-1, ETA and ETB receptors in breast carcinomas was also found to be indirectly related to disease-free survival time and overall survival. According to a study by Wülfing et al., patients who were ETA receptor positive, ETB receptor positive, and/or ET-1 positive had significantly decreased disease-free survival time and overall survival. For example, the mean disease-free survival time for ETA receptor positive patients was 74 (± 6) months and for ETA receptor negative patients was 90 (±4) months. Evidently, increased levels of endothelin and its receptors correlate to decreased disease-free survival time and overall survival.17

Research in Treatment for Prostate and Breast Cancer:

     Treatment for prostate cancer has been an enigma since the detection of the cancer. With the recent breakthrough discovery of endothelin receptor antagonists, however, there is hope for an effective and painless treatment method. The use of endothelin receptor antagonists as treatment can only be considered after failure of hormonal therapy, which is the primary method of care.18 The ETA receptor antagonists have been found to inhibit the effects of ET-1.19 The most significant of the agents under scrutiny is atrasentan, also known as ABT-627. This antagonist is an effective, orally bioavailable substance that is highly selective and designed to block the action of endothelin-1.19 Atrasentan shows promise of reducing bone formation and delaying the cancer’s progression in men who no longer respond to hormone therapy.20 Clinical trials with atrasentan have generally demonstrated decreases in the amount of pain, bone deposition, and bone metastases.19
Several clinical trials have been conducted to test the efficacy of atrasentan on men with prostate cancer. Dr. Michael Carducci, with his team at The Johns Hopkins Cancer Center, and Dr. Bernard Zonenberg, with his team from The Netherlands, performed Phase I trials on 22 men with adenocarcinoma. The trials were conducted for 28 days, without blindings or placebos. The results were in favor of atrasentan, with approximately 68% of the subjects experiencing regression in concentrations of prostate specific antigens and approximately 70% of the subjects experiencing less pain compared to the previous treatments. The most significant side effects were rhinitis, headache, asthenia, and peripheral edema.7

      Phase II trials, with double-blindings and placebos, were performed following the encouraging results of the Phase I trials. During these trials, a total of 244 men with prostate cancer received either a placebo, 2.5 mg of atrasentan, or 10.0 mg of atrasentan every day for a maximum of 196 days. In the subjects who received the placebo, the mean number of days before there was detectable clinical progression of the cancer was 129 days, and the mean number of days before there was a significant increase in prostate specific antigens was 71 days. In the subjects who received 2.5 mg of atrasentan, the mean number of days before there was detectable clinical progression of the cancer was 184 days, and the mean number of days before there was a significant increase in prostate specific antigens was 134 days. In the subjects who received the 10.0 mg of atrasentan, the mean number of days before there was detectable clinical progression of the cancer was 196 days, and the mean number of days before there was a significant increase in prostate specific antigens was 155 days. As in the Phase I trials, the side effects were rhinitis, headache, and peripheral edema.7 Clinical trials with atrasentan have shown good tolerability of the drug and the drug-related effects are relatively mild (Figure 4).20

      The direct correlation between the amounts of atrasentan administered and the prolongation of time before disease progression clearly suggests the beneficial effects that atrasentan can deliver. Currently, Phase III trials are being conducted; however, no one has yet to publish conclusions.21

     During each of the phase II trials of atrasentan, the osteoblastic activity of the bone was also observed. In the study, 147 men received a placebo, 135 men received 2.5mg of atrasentan, and 137 men received 10mg of atrasentan. The experimenters used markers, such as serum total and bone alkaline phosphatase, for bone deposition and collagen cross linked urine N-telopeptides, serum C-telopeptides, and urine deoxypyridinoline for bone reabsorption. The subjects who were receiving 10mg experienced no osteoblastic activity; however, those receiving a placebo experienced a 114% increase in bone alkaline phosphatase, which indicated increased osteoblastic activity.7

      One of the gene-targeted therapies in development is the use of Xinlay from Abbot Laboratories. This drug blocks the endothelin receptors, which will prevent the prostate cancer from metastasizing. In a 3 month study of approximately 1,100 patients, pain related to bone metastases decreased with minimal side effects.22

      Research in treatment using endothelin for breast cancer is currently in development. It has been suggested that targeting the effects of endothelins and their receptors on angiogenesis in the breast will have clinical significance. No specific drugs have been identified for this use.16

Conclusion

     ET-1 is a vasomotor regulator, cell proliferators, hormone producer, and tissue differentiator and developer. The overproduction of endothelin-1 in prostate and breast neoplasms leads to metastasis, angiogenesis, inhibition of apoptosis, and cancer cell proliferation. Many studies have been conducted to verify the prominence of the endothelin axis in these cancers. Atrasentan is one of the drugs considered for treatment after hormone therapy fails. Regulation of the production of endothelin is a significant objective of future research .

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