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BCRL Home » Research Projects » Breast Cancer Therapy by Differentiation Gene Activation

Breast Cancer Therapy by Differentiation Gene Activation

Dr. Irma H. Russo  

Dr. Irma H. Russo, together with Dr. Jose Russo, pioneered the hormone induced differentiation of the  breast as a preventive mechanism of cancer prevention.

 
Sporadic breast cancer is the fatal disease most frequently diagnosed in American women from all ethnic groups. It has been increasing at a rate of approximately 6% per year. Similar trends are observed worldwide, even in countries characterized by their low breast cancer incidence. Improved detection methods and diagnosis at an early stage have resulted in a decline in breast cancer mortality in the United States. Nearly one third of lymph node negative and more than one half of lymph node positive women that will eventually develop metastatic disease, as well as all women with metastatic disease from the time of diagnosis will die of breast cancer. The incurability of the disease, in association with the worldwide increase in incidence indicates that primary prevention is the ultimate goal for breast cancer control. However, the possibility of developing strategies for preventing the initiation of cancer are hindered by the multistep nature of the process, and the facts that only inheritance of cancer-predisposing genes, and radiation exposure at a young age have been identified as a mechanism or causal agent associated with cancer initiation. Current strategies to prevent breast cancer have focused on a unique feature of this disease, its endocrine, namely estrogen, dependence, which can be manipulated to control growth or prevent tumor development utilizing either selective estrogen receptor modulators (SERMs), such as tamoxifen, or aromatase inhibitors (AI's), such as Arimidex, Letrozole and Exemestane. However, these strategies are not widely acceptable to a majority of treated women who would not have developed breast cancer even if untreated. Therefore what is needed is to develop a new paradigm for breast cancer prevention.  

The new paradigm in breast cancer prevention

The new paradigm has emerged from epidemiological observations of a direct association of breast cancer risk to nulliparity and of protection conferred by an early first full term pregnancy. Why has this specific angle been chosen and not another? Because this paradigm illustrates a window of opportunity that nature offers for learning how a physiological event produces, in a significant percentage of women, a complete protection against cancer. This physiological event does not explain all the questions about breast cancer, but it provides a blueprint for a new paradigm in breast cancer prevention. The novelty of this paradigm does not germane from the knowledge that an early first full term pregnancy protects the breast against neoplastic transformation, but from studies that unveil the biological principle underlying the protection conferred by an early first full-term pregnancy and by demonstrating experimentally that it induces in the breast the expression of a specific signature that results from the completion of a cycle of this organ's differentiation driven by the reproductive process. This signature, in turn, is a biomarker associated with lifetime decreased breast cancer risk. More importantly, the biological principlehas been harnessed by demonstrating in an experimental model that a short treatment with human chorionic gonadotropin (hCG), a placental hormone secreted during pregnancy, induces the same genomic signature than pregnancy, inhibiting not only the initiation but also the progression of mammary carcinomas, stopping the development of early lesions, such as intraductal proliferations, and carcinomas in situ. These observations indicate that hCG administered for a very short period of time has significant potential as a chemopreventive agent, protecting the normal cell from becoming malignant. This new biological concept also implies that when the genomic signature of protection or refractoriness to carcinogenesis is acquired, the hormonal treatment with hCG is no longer required. This is a novel concept is in contraposition to the current knowledge that a chemopreventive agent needs to be given for a long period to suppress a metabolic pathway or abrogate the function of an organ.

Epidemiological and Clinical basis for the new strategy in prevention

Epidemiological and clinical evidence indicate that endocrinological and reproductive influences play major roles in breast cancer. It has long been known that the incidence of breast cancer is greater in nulliparous than in parous women. Changes in lifestyle that in turn influence the endocrinology of women have been observed during the last decades in American women, namely a progressive decrease in the age of menarche and a progressive increase in the age at which a woman bears her first child. The significance of these changes is highlighted by the reduction in breast cancer risk associated with late menarche and the completion of a full term pregnancy before age 24, with further reduction in the lifetime breast cancer risk as the number of pregnancies increases. Women who undergo their first full term pregnancy after age 30, on the other hand, appear to be at higher risk of breast cancer development than nulliparous women, suggesting that parity-induced protection against breast cancer is related to the timing of a first full-term pregnancy.Although pregnancy appears to have a dual effect on breast cancer risk, a transient increase (relative to nulliparous women) lasting 10-15 years, followed thereafter by a decreased risk, the protection conferred lasts a lifetime . Of interest is the fact that women from different countries and ethnic groups exhibit a similar degree of parity-induced protection from breast cancer, regardless of the endogenous incidence of this malignancy. This observation suggests that the reduction in breast cancer risk associated with early first full-term pregnancy does not result from factors specific to a particular environmental, genetic, or socioeconomic setting, but rather from an intrinsic effect of parity on the biology of the breast (which nevertheless may be modified by environmental, genetic, or other factors). These observations indicate that an early first full-term pregnancy modifies, through mechanisms still poorly understood, specific biological characteristics of the breast that result in a lifetime decreased risk of cancer development. This protection, nevertheless, is being attributed in great part to the induction of terminal differentiation of the mammary gland, a mechanisms that has been found to reduce the susceptibility of the mammary epithelium to carcinogenesis. These observations indicate that the terminally differentiated state of lactation should be reached for attaining protection, although other mechanisms have been proposed for the protective effect of early first full-term pregnancy, including the occurrence of sustained changes in the level or regulation of hormones that affect the breast. Regardless the intervening mechanism, the end result of the first pregnancy is a dramatic modification of the architecture of the breast. The direct association of breast cancer risk with nulliparity, as well as the protection afforded by early first full term pregnancy has been in great part explained by experimental studies. 

Data from Experimental animal studies.

The direct association of breast cancer risk with the prolongation in the period encompassed between menarche and the first full term pregnancy, as well as the protection afforded by pregnancy has been partially explained by experimental studies performed in laboratory animals. It has been demonstrated that mammary cancer in rodents can be induced with the polycyclic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) preferentially when the carcinogen is administered to young nulliparous females. Those females that have completed a full term pregnancy prior to carcinogen exposure fail to develop carcinomas. Altogether these studies have revealed that the susceptibility of the mammary gland to be transformed by a chemical carcinogen is modulated by specific biological conditions of the host and of the target organ. Tumor incidence and number of tumors per animal, which are the biological endpoints when evaluating tumorigenic response, are maximal when the carcinogen is administered to young but cycling virgin rats. Cancer incidence is directly proportional to the number of terminal end buds (TEBs) that are at their peak of cell proliferation. Stimulation of the development and differentiation of the gland, resulting in profuse lobular development and depression of DNA synthesis, such as it occurs during pregnancy, or after completion of a 21 day-treatment of virgin rats with hCG, reduce the susceptibility of the mammary epithelium to be transformed by the carcinogen. The reduction in cancer incidence is permanent, as demonstrated by the similar degree of reduction when DMBA is administered after a delay of 21, 42, or 63 days after termination of hCG treatment.

Pregnancy alone or followed by lactation, induces in the mammary gland a permanent protective effect from chemically induced carcinogenesis, since administration of a carcinogen to parous rats when the glands have regressed to a resting stage either fails to induce carcinomas or considerably lowers their incidence, whereas mammary glands showing gestational or lactational hyperplasia are moderately refractory to DMBA induced carcinogenesis. This indicates that it is not the transient hormonal status occurring during pregnancy and lactation that protects the mammary gland, but the permanent changes induced in the gland structure and in the biological properties of the glandular epithelium by the reproductive phenomenon.

The observation that pregnancy before carcinogen administration seems to be the only truly protective factor in chemically-induced mammary gland carcinogenesis, suggests that placental hormones play an important role inmammary growth and development during pregnancy. The main placental hormone, human chorionic gonadotropin (hCG) has a stimulatory effect on the mammary gland when administered exogenously, producing either a gestational or a lactational type of mammary development that considerably reduces the incidence of tumors,. The fact that the hormonal changes of pregnancy accelerate DMBA-induced mammary tumor growth when mating occurs after carcinogen administration indicates that the most important event in determining the role that this hormone plays in either preventing initiation or in promoting tumor growth is the sequence in which it reaches the mammary gland.

It has been demonstrated that the inhibitory effect of pregnancy on mammary cancer initiation is mediated by hCG, since virgin rats treated for 21 days with a daily intraperitoneal injection of this hormone prior to carcinogen administration exhibit a dose-related reduction in tumor incidence and number of tumors per animal. This phenomenon is in great part mediated by the induction of mammary gland differentiation, inhibition of cell proliferation, increase in the DNA repair capabilities of the mammary epithelium, decrease binding of the carcinogen to the DNA, and activation of genes controlling programmed cell death (PCD). The activation of these genes by hCG is of great relevance because PCD is a physiological and phylogenetically conserved form of active cell death (or apoptosis) that has been associated with specific phases of development that control cell proliferation and differentiation.

Data from Clinical Studies

Based on preclinical data that had demonstrated that hCG treatment of virgin rats prevented the initiation and inhibited the progression of DMBA-induced mammary carcinomas, the effect of hCG on primary breast cancer in post-menopausal patients was evaluated. In a double-blind, placebo-controlled study, 25 post-menopausal women with primary operable breast cancer (T1-T3) whose diagnosis was made by core biopsy performed on day 0, received on alternate days for 2 weeks intramuscular injections of either r-hCG (recombinant hCG) (500 g); n=20) or placebo (n=5). Surgery (mastectomy or lumpectomy) was performed on day 15. The tumor tissue obtained in the initial core biopsy and that removed at the time of therapeutic surgery were evaluated to determine the rate of cell proliferation, or proliferative (Ki67) index, inhibin immunoreactivity, and percentage of cells positive for estrogen (ER) and progesterone receptors (PR). The most remarkable effects attributed to this two-week treatment were a significant reduction in Ki67 index from 18% in the initial biopsy to 4% in the mastectomy/lumpectomy specimens (p<0.00006), and increased synthesis of inhibin.

Serum hormonal levels were those characteristic of post-menopausal women, and remained unchanged during and after the treatment, except for elevation in hCG levels during treatment. Hormone administration was well tolerated by all patients, and no local or systemic side effects were reported at any time. The data clearly indicated that hCG is an inhibitor of cell proliferation independently of ovarian function (postmenopausal women) and independent of the estrogen and progesterone receptor status of the host tissue. In addition, the data indicates that the recombinant form of this hormone does not affect the hormonal milieu of the patient.

 

Pregnancy and hCG induce permanent genomic imprinting or a specific signature of protection

The genomic signature of the mammary gland induced in virgin animals by exogenous administration of hCG is similar to that induced by pregnancy, and those specific genomic profiles are still manifested by 42 days post termination of treatment. The importance of these specific signatures is highlighted by the fact that administration of carcinogen to hCG-treated or control virgin rats whose mammary glands appear morphologically similar will induce a markedly different tumorigenic response, supporting the concept that the differentiation induced by hCG is expressed at the genomic level, and results in a shift of the susceptible stem cell 1 to a refractory stem cell 2. The permanence of these changes, in turn, makes them ideal surrogate markers for the evaluation of hCG effect as a breast cancer preventive agent.

Conclusion

Breast cancer originates in the undifferentiated terminal duct of the Lob 1 that contains stem cells (Stem cell 1) has been attributed to its high rates of cell proliferation and of carcinogen binding to the DNA and low reparative activity. The hormonal milieu of an early full term pregnancy or hCG treatment induces lobular development, completing the cycle of differentiation of the breast. This process induces a specific genomic signature in the mammary gland that is represented by the Stem cell 2 epithelium remains capable of responding with proliferation to given stimuli, such as a new pregnancy. The stem cell 2 is able to metabolize the carcinogen and repair the induced DNA damage more efficiently than the stem cell 1, as it has been demonstrated in the rodent experimental system. There is also evidence that hCG has an effect in the cancer cell by further the differentiation pattern. The finding that differentiation is a powerful inhibitor of cancer initiation provides a strong rationale for identifying the genes that control this process. The basic biological concept is that pregnancy, or hCG, shifts the stem cell 1 to the stem cell 2 that is refractory to carcinogenesis.

Future Perspective

The mechanisms discussed above play a role in the protection exerted by hCG from chemically induced carcinogenesis, and might be even involved in the life-time reduction in breast cancer risk induced in women by full term and multiple pregnancies. The implications of these observations are two-fold: on one hand, they indicate that hCG, like pregnancy, may induce early genomic changes that control the progression of the differentiation pathway, and that these changes are permanently imprinted in the genome, regulating the long-lasting refractoriness to carcinogenesis. The permanence of these changes, in turn, makes them ideal surrogate markers of hCG effect in the evaluation of this hormone as a breast cancer preventive agent.

References