IGF-II and cathespin D in tumor growth and metastasis

Institution: Loma Linda University
Investigator(s): Daisy De Leon, Ph.D. -
Award Cycle: 1996 (Cycle II) Grant #: 2KB-0059 Award: $353,776
Award Type: New Investigator Awards
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1996)
Many women die as a result of metastasis, the process by which tumor cells dissociate from the initial tumor and establish a new tumor at a different site. Currently, the most valuable predictor of breast tumor metastasis is the presence of cancer cells in lymph nodes. Nevertheless, new tumors reappear in 30% of patents with no cancer cells in the lymph nodes. Thus, more accurate prognostic indicators are needed. Our project is designed to investigate the basic biology of how three proteins interact (IGF-II, IGF-II receptor and cathepsin D) in breast cancer tumor growth and metastasis. Our proposal is relevant to the understanding of breast cancer development and metastasis, the major cause of death in breast cancer patients and an important BCRP priority area.

Cathepsin D appears to be a protein involved in metastasis. Several studies in breast cancer patients have demonstrated that high levels of cathepsin D correlated with significantly reduced cancer- free intervals after treatment. When studied by "immunocytochemistry" (the standard clinical laboratory assay) the correlation of cathepsin D and metastases have provided conflicting evidence. Thus, further studies with more precise assays for cathepsin D activity are necessary. Preliminary results suggest that high levels of "active" cathepsin D in breast tumors are associated with the risk of metastasis.

The present study seeks to validate this observation. IGF-II is a protein that stimulates cell growth and cathepsin D production. Both IGF-II and cathepsin D are secreted by breast cancer tumors and bind the IGF-II receptor. Despite distinct binding sites, IGF-II binding can affect cathepsin D binding to the IGF-II receptor. Thus, we believe that increased concentrations of IGF-II compete with cathepsin D for the IGF-II receptor; to restore the balance, increased secretion of cathepsin D occurs. We developed a breast cancer cell system that confirmed the theory and demonstrated how IGF-II blocked the transport of cathepsin D, increasing its secretion.

In summary, this proposal is designed to demonstrate that interactions between IGF-II and cathepsin D with the IGF-II receptors increase cathepsin D secretion, resulting in tumor growth and metastasis. To test this, we will determine whether: (1) IGF-II expression/cathepsin D secretion increases tumor growth and metastasis; and (2) Higher levels of specific forms of cathepsin D and IGF-II are found in breast tumor tissues than in normal breast tissues and are associated with metastasis. Expression of IGF-II and cathepsin D in paired normal and tumor tissue from breast cancer patients will be studied. Elucidation of the regulatory mechanism(s) in which these proteins participate and their interactions with factors contributing to the progression of breast cancer may have direct applications for improvements in patient treatment and follow-up.

Final Report (1999)
There is an urgent need to identify markers that can predict the risk of tumor spread (metastasis) in breast cancer patients. Cathepsin D is a protein linked with decreased breast cancer patient survival that may be a great tool to identify patients at a higher risk of metastasis. However, conflicting reports regarding the usefulness of this protein and the lack of knowledge regarding the cause of its release from breast cancer tumors has limited its use in patient prognosis. Our BCRP research project was design to investigate two critical questions: 1) what causes the release of cathepsin D from breast cancer cells? And 2) Can we use cathepsin D as a prognostic factor for breast cancer metastasis?

To answer these questions, we first developed a breast cancer cell model to determine whether the release of cathepsin D was related to the production of a protein known as IGF II. We also examined paired breast cancer tissues by two different cathepsin D assays to investigate if this protein was increased in tumors and how it correlated to tumor recurrence and metastasis. Tumor models in NUDE and SCID mouse were also developed and we demonstrated that tumors producing proIGF II grow faster and independent of estrogen. No tumors were able to grow when animals were injected with parental breast cancer cells or cells producing mature IGF II. This data demonstrates that proIGF II confers breast cancer cells with a growth advantage. Our studies demonstrated that IGF II increases the release of cathepsin D. The production of IGF II (a potent cell growth factor) interferes with cathepsin D storage process inside breast cancer cells causing cathepsin D to be released. Of great interest is the fact that the production of IGF II alters cathepsin D in cancer cells with and without estrogen receptors. Furthermore, production of IGF II by normal breast cells also alters the secretion of cathepsin D. Thus, our studies provide direct evidence correlating IGF II with cathepsin D secretion and present the first direct evidence of this mechanism in a human cell model. Since the increase of cathepsin D is being proposed to be associated to metastasis, we analyzed this protein by two different methods. Our results revealed that of the 5 6 different forms of cathepsin D, only one form was increased in breast cancer tissues. This difference cannot be assessed by current standard histological techniques used in clinical laboratories, thus explaining the conflicting reports from different laboratories.

We are at present collecting the retrospective patient data to determine its significance for prognosis. The potential use of cathepsin D, and possibly IGF II, to predict metastasis will have a significant impact in breast cancer patient treatment. Given the current lack of such markers' our results represent a much needed advancement to improve breast cancer patient survival and follow up.

Symposium Abstract (2005)
PI: Daisy D De Leon Co-PI: Carlos Garberoglio, MD

The disparity in survival among African American (AA) women affected with breast cancer is associated with poorly known clinical and pathological characteristics. A comprehensive approach to understand the biological factors associated with poorer outcomes among AA patients is urgently needed. Our laboratory is addressing this need by investigating how IGF-II and cathepsin D (CD) are associated with breast cancer patientís outcome. CD and IGF-II are proteins secreted by breast tumors that are aggressive and metastatic, and high levels of these factors in blood from breast cancer patients correlates with poor survival. Utilizing breast tissues from the Loma Linda University tumor bank, we analyzed by Western blotting the expression of IGF-II and CD. We also analyzed tissue slides made from these same patients to detect the presence of IGF-II and CD. Our preliminary studies showed that significantly higher levels of CD are present in paired normal/tumor tissues from AA patients as compared to Caucasian patients. When tumor tissues where compared, CD was two-fold (2x) higher in tissues from AA patients as compared to tissues from Caucasian patients. Surprisingly, CD levels in normal tissues from AA patients were six-fold higher than normal tissues froth Caucasian patients. Examination of the breast tissues of these patients under the microscope revealed that "normal tissues" from AA patients contained abundant fibrocystic changes. This is a significant observation since it is well known that women with fibrocystic changes in their, breast have a higher risk of developing breast cancer. Both, IGF-II and CD were detected in normal tissues with fibrocystic changes analyzed in our study. Normal epithelial tissue does not contain IGF-II nor does it secrete CD. Thus, expression of IGF-II and secretion of CD by fibrocystic tissues may represent initial markers of breast cancer development. Of note, we examined the secretion of GPII and CD from "normal" mammary cells established from fibrocystic tissue of a healthy woman that had a breast reduction procedure. Those cells known as NICF-10 cells secreted high levels of CD and also secreted IGF-II. These observations are exciting in view of the soon to be published data from our laboratory (in part funded by CBCRP) demonstrating that 1) cells secreting IGF-II and CD grow faster, 2) tumors established from human breast cancer cells that produce IGF-II and secrete CD can develop and metastasize without the need of estrogen in a mouse model.

Correlation of CD and IGF-II with disease progression is a promising development that can benefit breast cancer patients in general by improving diagnosis and follow up treatment. The present study provide important insight in understanding how the differential expression of biological factors such as IGF-II and CD can account for the survival disparity observed among AA breast cancer patients. More comprehensive studies to assess how these factors may offer a unique therapeutic target for a molecular medicine approach to the treatment and prevention of breast cancer among AA are needed. Identifying markers of poorer prognosis is vital to increase survival rates and eliminate the mortality burden afflicting AA patients.

Reversal of Cathepsin D Routing Modulation in MCF-7 Breast Cancer Cells expressing antisense Insulin- like Growth Factor II (IGF-II)
Periodical:Hormone and Metabolic Research
Index Medicus: Horm Metab Res
Authors: De Leon, D.D., Issa, N.N., Nainani, S.B. and Asmeron, Y.
Yr: 1999 Vol: 31 Nbr: Abs: Pg:142-147