Lucy Seiple

Writer’s comment: When I first received the assignment to write a literature review paper for my English 104E (Scientific Writing) class, I was intimidated by the task and unsure of what I would write about. Searching the databases eventually led me to the topic of angiogenesis in tumors. Having never read a scientific journal article before taking this class, I found writing the review essay to be a real challenge. I had to read numerous articles, decipher the complicated language, understand the topic, and extract only the relevant information from each paper. Through this process, though, I learned a great deal, not only about angiogenesis but also about researching and writing. The assignment was daunting at first, but the reading and writing skills I gained in completing it will certainly benefit me in my future.
- Lucy Seiple

Instructor’s comment: The review paper Lucy Seiple prepared for English 104E: Scientific Writing accomplishes just what a strong review ought to do: It informs us about the state of current research on her subject, breast cancer in this case, a matter of considerable public significance. Specifically, Lucy focuses her attention on studies devoted to understanding a factor that aids in the formation of the blood vessels that nourish tumors, and she does an excellent job of synthesizing relevant findings from this promising work.
- Sondra Reid, English Department



Introduction

Because the incidence of breast cancer is on the rise in North America, current research is focusing on successfully treating breast cancer and preventing relapses. Nearly two decades ago, researchers discovered that angiogenesis, the formation of new blood vessels, was vital for the growth and metastasis of tumors, which require a blood supply for nutrients and oxygen. Disrupting angiogenesis has therefore been targeted as a promising way to fight breast cancer.
     Many factors necessary for angiogenesis in breast cancer tumors to occur have been identified. Of these angiogenic factors, vascular endothelial growth factor (VEGF) appears to play a crucial role in tumor angiogenesis (Aonuma et al., 1999; Borgstrom et al., 1999; Kranz et al., 1999; Kurebayashi et al., 1999; Lewin et al., 1999; Ruohola et al., 1999; Zhai et al., 1999). Elevated levels of VEGF have been correlated with the advancement of angiogenesis and with early relapse after successful cancer treatment (Borgstrom et al., 1999). It has also been proposed that VEGF levels as well as vessel density could be potential prognostic factors for breast cancer. In view of its apparent role in tumor angiogenesis, studies have attempted to detect mechanisms for upregulation of VEGF and possible methods for inhibiting the protein as a means of treating breast cancer.

VEGF and Its Role in Tumor Angiogenesis

     VEGF is an important cytokine that is produced by endothelial cells. While it was originally discovered in tumor tissue, its primary function in normal tissue is better understood. VEGF binds a protein receptor in the endothelium vessels, initiating a signal cascade that ultimately results in neovascularization. The VEGF family includes four different forms of the VEGF protein: VEGF-A, -B, -C, and -D. Each form is expressed at different levels in both normal and cancerous tissue (Kurebayashi et al., 1999).
     VEGF exists in relatively low amounts in normal adult tissue. Its high expression in tumor tissue led researchers to identify it as a possible key factor in tumor angiogenesis. VEGF and its primary receptor, KDR, were found to be expressed at far higher levels in cancerous samples when compared with the normal tissue surrounding the tumor (Kranz et al., 1999). Expression levels of the different forms in tumor tissue have been examined as well. Using RT-PCR and cancerous breast tissue taken from node-positive and node-negative samples, researchers identified elevated levels of all four forms of VEGF in all of the tumor tissues when compared with normal tissue. Levels of VEGF-C and -D, however, were significantly higher in node-positive than in node-negative tissue. These results imply that all four forms of VEGF have enhanced expression in cancerous tissue, but that isoforms C and D play a particularly important role in the spread of cancerous cells throughout the body (Kurebayashi et al., 1999).
     Although the elevated level of VEGF in breast cancer cells is evidence of its involvement in angiogenesis of breast cancer tumors, other protein factors have also been implicated in angiogenesis. Most prominent of those is basic fibroblast growth factor (bFGF). To provide evidence that VEGF is the primary factor in malignant growth versus bFGF, researchers transformed tumor cells containing different levels of bFGF with VEGF and observed their angiogenic potentials. Cells with low levels of bFGF exhibited an increased rate of angiogenesis once transfected with VEGF. Cells with higher levels of bFGF exhibited a faster tumor growth rate, indicating that bFGF does aid in the rate of angiogenesis. However, when researchers transfected cells with VEGF that had no detectable bFGF, they still observed rapid tumor growth (Aonuma et al., 1999). These results strongly suggest that while bFGF is an important contributor to the rate of tumor growth, VEGF is the predominant factor in tumor angiogenesis.

Mechanisms of Upregulation of VEGF in Breast Tumor Cells

     While it is now accepted that VEGF is overexpressed in tumor cells, little is understood about the mechanisms of upregulation. Several different mechanisms have been suggested. One proposal is that the tumor makes and releases a substance that leads to an enhancement of VEGF and its receptor. Studies have shown that there is an increase in the number of VEGF receptors in the vessels near a tumor, supporting the theory of a tumor-induced upregulator. Other studies have targeted hypoxia (lack of oxygen) and such steroid hormones as estrogen as possible upregulators (Kranz et al., 1999).
     Researchers observed a significant increase in the levels of VEGF when they treated breast cancer cells with cobalt chloride, a compound that imitates the effects of hypoxia by displacing oxygen from hemoglobin. This observation provides evidence that hypoxia is an important upregulator of VEGF (Ruohola et al., 1999). When the tumor cells are deprived of oxygen, cells are signaled to enhance expression of VEGF and KDR, resulting in increased angiogenesis within the tumor.
     Although steroid hormones are involved in the development of mammary glands, it has not been shown how these hormones affect VEGF expression in breast tissue. To study this, Ruohola et al. (1999) examined the effects of steroid hormones on the expression of VEGF. They treated cancer cells with 17-b-estradiol (a potent estrogen) and observed a dramatic increase in the levels of VEGF transcripts within 1 hour of treatment. They also discovered that the mRNA was stabilized in the presence of the 17-b-estradiol, leading to a prolonged half-life. By performing a Western blot, they observed that the increase in VEGF mRNA correlated with an increase in the expression of the protein. Results from this analysis showed that expression of the protein did coincide with the increased levels of mRNA and confirmed that 17-b-estradiol upregulates expression of VEGF in cancer cells (Ruohola et al., 1999).

Inhibition of VEGF as a Means of Breast Cancer Treatment

Antiestrogens
     Because the steroid hormone estrogen has been identified as an upregulator of VEGF, researchers examined whether using antiestrogens in treating breast cancer would stop tumor growth by inhibiting angiogenesis. In fact, two such antiestrogens, tamoxifen and toremifene, are currently being used in breast cancer treatment for their ability to inhibit cell proliferation and to induce apoptosis. Ruohola et al. (1999) examined whether these drugs would also limit angiogenesis as expected, based on their upregulation studies. To their surprise, however, these two antiestrogens did not inhibit the expression of VEGF. Instead, antiestrogens increased the levels of VEGF mRNA in the cells. These contradictory results imply that antiestrogens are also factors of VEGF upregulation. Although their ability to stop cell proliferation has made them viable treatments for breast tumors, antiestrogens are potentially providing the tumor with a greater blood supply, an undesired result.

Anti-Vascular Endothelial Growth Factor Blocking Antibody
     Other research has focused on using antibodies to effectively block the VEGF receptors, thereby inhibiting VEGF activity and stopping tumor angiogenesis (Borgstrom et al., 1999). By using antibodies against the receptor, researchers were able to specifically inhibit VEGF while leaving other angiogenic factors intact. Borgstrom et al. (1999) looked at the effectiveness of an anti-VEGF antibody, A4.6.1, as an inhibitor of angiogenesis. They treated cancer cells with A4.6.1 and observed complete inhibition of angiogenesis and a cessation of tumor growth, showing that inhibiting VEGF alone was sufficient to stop the spread and growth of the tumor. In addition, these results strengthened the argument that VEGF is the main protein necessary for tumor angiogenesis and that using antibodies against the VEGF receptor could be an effective method for treating breast cancer.

Vascular Endothelial Growth Inhibitor (VEGI)
     Another novel approach to inhibiting VEGF began with the discovery of a protein, produced in vivo, that inhibits VEGF. Understanding that angiogenesis is a relatively infrequent event in normal endothelial tissue, researchers believed that there must be a factor that stops angiogenesis when it is not necessary in normal tissue. This factor was found and named vascular endothelial growth inhibitor (VEGI) (Zhai et al., 1999). Endothelial cells in normal tissue express VEGI to inhibit angiogenesis. However, VEGI is not expressed in endothelial cells within tumor tissue. Zhai et al. (1999) added VEGI to breast cancer cells and observed marked inhibition of tumor growth by suppression of neovascularization, suggesting that VEGI is a negative regulator of angiogenesis and may also be a successful approach to breast cancer treatment.

Conclusion

     VEGF has been identified as a primary factor in angiogenesis in tumor cells. Its involvement has been linked to a number of different cancers, including breast cancer (Borgstrom et al., 1999; Kranz et al., 1999; Ruohola et al., 1999; Zhai et al., 1999). With higher expression of VEGF, there is an enhancement of angiogenesis and a corresponding increase in vessel density, which tends to correlate with more extreme and terminal cancers (Kranz et al., 1999). Understanding the mechanisms of upregulation and inhibition of VEGF could potentially lead to its becoming an important factor in diagnosis and treatment of breast cancer. By assessing levels of VEGF, physicians might better diagnose their patients and provide appropriate care.


Literature Cited

Aonuma, M., Saeki, Y., Akimoto, T., Nakayama, Y., Nishikawa, K., Shibuya, M., Tanaka, N.G. (1999). Vascular endothelial growth factor overproduced by tumor cells acts predominantly as a potent angiogenic factor contributing to malignant progression. International Journal of Experimental Pathology 80, 271-–281.

Borgstrom, P., Gold, D.P., Hillan, K.J., Ferrara, N. (1999). Importance of VEGF for breast cancer angiogenesis in vivo: Implications from intravital microscopy of combination treatments with anti-VEGF neutralizing monoclonal antibody and doxorubicin. Anticancer Research 19, 4203–4214.

Kranz, A., Mattfeldt, T., Waltenberger, J. (1999). Molecular mediators of tumor angiogenesis: Enhanced expression and activation of vascular endothelial growth factor receptor KDR in primary breast cancer. International Journal of Cancer 84, 293–298.

Kurebayashi, J., Otsuki, T., Kunisue, H., Mikam,i Y., Tanaka, K., Yamamoto, S., Sonoo, H. (1999). Expression of vascular endothelial growth factor (VEGF) family members in breast cancer. Japanese Journal of Cancer Research 90, 977–981.

Lewin, M., Bredow, S., Sergeyev, N., Marecos, E., Bogdanov, A., Weissleder, R. (1999). In vivo assessment of vascular endothelial growth factor-induced angiogenesis. International Journal of Cancer 83, 798–802.

Ruohola, J.K., Valve, E.M., Karkkainen, M.J., Joukov, V., ,Alitalo, K., Härkönen, P.L. (1999). Vascular endothelial growth factors are differentially regulated by steroid hormones and antiestrogens in breast cancer cells. Molecular and Cellular Endocrinology 149, 29–40.

Zhai, Y., Yu, J., Iruela-Arispe, L., Huang, W.Q., Wang, Z., Hayes, A.J., Lu, J., Jiang, G., Rojas, L., Lippman, M.E., Ni, J., Yu, G.L., Li, L.Y. (1999). Inhibition of angiogenesis and breast cancer xenograft tumor growth by VEGI, a novel cytokine of the TNF superfamily. International Journal of Cancer 82, 131–136.