Progesterone Metabolites Regulate Breast Cell Tumors
Estrogens and progesterone have long been considered to play cardinal roles in breast cancer, and therefore, expressions of ER and PR are widely used as indicators of hormonal dependency and as determinants for current hormone-based treatments. The relevance of the steroid receptors is highlighted by observations that ER/PR-negative breast cancer cells such as MDA-MB-231, which normally do not respond to estrogen and progesterone, can become responsive, both in vitro and in vivo, if they are transfected with the receptors. Thus, the prevailing theory of hormonal regulation of breast cancer, as well as hormone-based therapies, revolves around estrogen and/or progesterone and ER/PR-positive breast cells and tumors. However, a large percentage of breast tumors are ER and/or PR negative and therefore are not explained by the actions of estrogen and/or progesterone. Not only do these "receptor-negative" breast cancers fail to benefit from current hormonal therapies, but they also generally exhibit more-aggressive biologic behaviors and poorer prognosis than the receptor-positive ones. The results of the studies reported here show for the first time that the progesterone metabolites, 5αP and 3αHP, act as hormones that regulate ER/PR-negative breast tumor formation, growth, and regression. The onset of the ER/PR-negative human breast cell tumors in mice was considerably accelerated, and the growth significantly stimulated, by just one or two applications of 5αP. In contrast, 3αHP retarded onset of tumor formation, suppressed tumor growth, and inhibited or regressed existing 5αP-induced tumors. When both hormones were administered simultaneously, the effects of one were abrogated by the effects of the other. The current in vivo demonstrations, from five separate experiments, of the opposing actions of 5αP and 3αHP on tumorigenesis and tumor growth extend the previous findings, which showed opposing in vitro cancer-regulatory actions of these progesterone metabolites on receptor-negative (MDA-MB-231, MCF-10A) as well as receptor-positive (MCF-7, T47D, ZR-75-1) breast cell lines.
The response of ER/PR-negative breast cells to the progesterone metabolites can be explained by the presence of specific high-affinity receptors for 5αP (5αPR) and 3αHP (3αHPR). The 5αPR and 3αHPR (which are associated with the plasma membranes of both ER/PR-positive and ER/PR-negative cells) are distinct from each other and from known ER, PR, androgen, and corticosteroid receptors, and lack affinity for other steroids, such as progesterone, estrogen, androgens, corticosteroids, and other progesterone metabolites. Levels of 5αPR are upregulated by 5αP itself and estradiol, and downregulated by 3αHP in both ER/PR-positive and -negative cells. The mechanisms of action resulting in the opposing effects of these two hormones appear to involve cell-signaling pathways associated with the plasma membrane receptors, as well as altered gene expression. Indications are that 5αP acts via the surface receptor-linked mitogen-activated protein kinase (MAPK; Erk1/2) pathway; 5αP significantly stimulates activation of Erk1/2, increases the Bcl-2/Bax expression ratio and actin depolymerization, and decreases expression of actin and adhesion plaque-associated vinculin, resulting in decreased apoptosis and increased mitosis and cell detachment. Conversely, 3αHP appears to suppress protein kinase C (PKC), phospholipase C (PLC), Ca mobilization (unpublished observations), and the Bcl-2/Bax expression ratio, and increases expression of the cell-cycle inhibitor p21, resulting in increased apoptosis and decreased proliferation and detachment of breast cell lines. In pituitary cells, 3αHP also has been shown to inhibit a plasma membrane-associated PKC, PLC, Ca cell-signaling pathway.
The results of the studies reported here not only show that 5αP and 3αHP have opposing effects on initiation and growth of ER/PR-negative human breast tumors, but also provide in vivo evidence of the marked changes in the relative concentrations of these hormones in the tumor microenvironment. Whereas serum from control mice, in which implanted human breast cells had not developed into tumors, contained about equal concentrations of 5αP and 3αHP, serum from mice with tumors had significantly more 5αP than 3αHP. Because hormones had not been administered to these mice, the higher 5αP/3αHP ratio in serum from tumor-bearing mice can reasonably be expected to have resulted from the tumors, which on average had about threefold higher concentrations of 5αP than the respective sera, and >10-fold higher 5αP than 3αHP levels. Previous in vitro metabolism studies showed that human breast tumor tissues convert significantly more progesterone to 5α-pregnanes like 5αP and less to 4-pregnenes like 3αHP than do paired normal (nontumorous) tissues and that these differences correlated with significantly higher 5α-reductase gene (SRD5A1, SRD5A2) and lower 3α(20α)-HSO gene (AKR1C1, AKR1C2, AKR1C3) expression in tumor tissues. Similar differences in progesterone metabolism and enzyme gene expressions were observed between tumorigenic and nontumorigenic breast cell lines. In addition to the ability to convert progesterone to active cancer-regulating hormones, breast carcinomas are able to synthesize progesterone, which could account for its relatively high concentrations in the xenograft tumors reported here, and indicate an in situ supply of the biosynthetic precursor of 5αP and 3αHP.
The significant concentrations of 5αP and 3αHP, and particularly the high 5αP/3αHP ratios, in the MDA-MB-231 xenograft tumors, emphasize the potential importance of the microenvironment within breast tissue where the biologic actions occur. The role of the microenvironment in changing the expression of regulatory factors such as metabolizing enzymes, receptors, cytoskeletal and adhesion molecules, and growth promoters/inhibitors and in epigenetic alterations has been extensively reviewed. The current findings, along with the previous in vitro studies, suggest that the relative concentrations of 5αP and 3αHP in the breast microenvironment constitute important autocrine/paracrine determinants not only for tumorigenesis but also for potential regression of tumors and the maintenance of normalcy of ER/PR-negative breast cells/tissues. Figure 8 provides a summary of opposing biologic actions and proposed mechanisms of action of the progesterone metabolites, 5αP and 3αHP, in promoting neoplasia and tumorigenesis, as well as in maintaining normalcy in ER/PR-negative human breast cells. Evidence presented here shows that a high concentration of 5αP, relative to 3αHP in the microenvironment, promotes initiation and growth of tumors, whereas a higher concentration of 3αHP, relative to 5αP, suppresses tumorigenesis and promotes normalcy. Previous evidence indicates that these opposing effects of 5αP and 3αHP are propagated via the opposing actions of the hormones on cell proliferation (mitosis, apoptosis), adhesion, cell cycle, regulatory and signaling molecules, and gene expression after binding to specific receptors.
(Enlarge Image)
Figure 8.
Summary of the opposing autocrine/paracrine effects of the progesterone metabolites, 5αP and 3αHP, in a stylized ER/PR-negative human breast cell. Evidence presented here shows that a high concentration of 5αP relative to 3αHP, in the microenvironment, promotes initiation and growth of ER/PR-negative human breast cell tumors, whereas a higher concentration of 3αHP, relative to 5αP, suppresses tumorigenesis and promotes normalcy. Progesterone is converted to 3αHP and 5αP in breast cells. Tumorigenic and tumor cells convert more progesterone to 5αP and less to 3αHP than do normal cells. The steroids, being lipophylic, are able to pass out of cells and result in a concentration buildup in the microenvironment. The result is a significant increase in the 5αP-to-3αHP concentration ratio in the microenvironment of tumorigenic cells and within tumorous tissues in comparison with normal (nontumorous) breasts. 3αHP and 5αP bind to specific receptors on the plasma membrane linked to signaling pathways involving PKC, phospholipase C, and Ca mobilization (3αHP) and MAPK/Erk1/2 (5αP) and to modulators of gene expression. The cancer-inhibiting actions of 3αHP result in decreased proliferation and detachment of cells, increased apoptosis, and suppression of tumor initiation and growth. The cancer-promoting actions of 5αP have the opposite effects and result in stimulation of tumorigenesis and tumor growth. The evidence suggests that high concentrations of 5αP relative to 3αHP in the microenvironment will promote progression toward neoplasia and tumorigenesis, whereas a low 5αP-to-3αHP concentration ratio favors maintenance of the normal state.
How might higher levels of either 5αP or 3αHP in the serum due to the steroid implants have, respectively, initiated/promoted or suppressed xenograft tumorigenesis? The 5αP and 3αHP treatments, consisting of suspensions placed subcutaneously in the nape of the neck, resulted in elevated serum levels of either hormone, which persisted for about 2 to 3 weeks after the last injection. Because the depots were not far removed from the site of the cell implants, lymph drainage may have resulted in significantly higher concentrations of each applied hormone in the immediate vicinity of the implanted human cells. Conceivably, in the 5αP-treated mice, the induced elevation of 5αP levels, relative to 3αHP, in the microenvironment of the human cell implants, could have exerted procancer actions that initiated tumorigenesis. Because 5α-reductase and 5αPR levels are upregulated by 5αP, the in situ production and paracrine/autocrine actions of locally elevated 5αP could then have autoenhanced hormone-receptor interaction and the resulting stimulation of tumor growth, as illustrated in Figure 8. In a like manner, in the 3αHP-treated mice, the elevated 3αHP levels, relative to 5αP, in the microenvironment could have opposed progression to xenograft neoplasia by its inherent anticancer actions and the suppression of 5αP synthesis and 5αPR expression. By extension, in an intact human breast, local changes in relative concentrations of 5αP and 3αHP (that is, changes in the 5αP/3αHP ratio) resulting from selective up- or downregulation of progesterone-metabolizing enzymes induced by microenvironmental triggers could determine ER/PR-negative breast cell progression to tumor initiation and growth or maintenance of normalcy. Because only small changes in enzyme activity/expression are needed to result in significant local concentration changes, either a slight elevation of 5α-reductase, or a reduction of 3α-HSO, in one or more cells could lead to an increase in the ratio of 5αP/3αHP in the immediate intra- and extracellular environment. Conversely, processes that result in higher levels of cancer-suppressing 3αHP (and consequently lower 5αP/3αHP ratios) could ensure maintenance of normalcy.
Because in vitro studies have shown that both ER- and/or PR-negative and -positive breast cells respond in a stimulatory and inhibitory fashion, respectively, to 5αP and 3αHP, and have 5αPR and 3αHPR, the present results also may have implications for the substantial numbers (20% to 40%) of ER/PR-positive patients who fail to respond to suppression of estrogen and/or progesterone levels or actions, as well as for those receptor-positive tumors that do respond to hormonal therapies. In this regard, the opposing actions of the progesterone metabolites also appear to exert some control over the estrogen-regulated effects on breast cancer by their ability to modulate ER numbers in ER-positive cells. Furthermore, because both tumorigenic and normal (nontumorigenic) breast cells respond to the opposing actions of 5αP and 3αHP, the relative concentrations of the progesterone metabolites in the microenvironment may also play a role in maintaining normalcy of breast tissues in general, regardless of ER/PR status.
Discussion
Estrogens and progesterone have long been considered to play cardinal roles in breast cancer, and therefore, expressions of ER and PR are widely used as indicators of hormonal dependency and as determinants for current hormone-based treatments. The relevance of the steroid receptors is highlighted by observations that ER/PR-negative breast cancer cells such as MDA-MB-231, which normally do not respond to estrogen and progesterone, can become responsive, both in vitro and in vivo, if they are transfected with the receptors. Thus, the prevailing theory of hormonal regulation of breast cancer, as well as hormone-based therapies, revolves around estrogen and/or progesterone and ER/PR-positive breast cells and tumors. However, a large percentage of breast tumors are ER and/or PR negative and therefore are not explained by the actions of estrogen and/or progesterone. Not only do these "receptor-negative" breast cancers fail to benefit from current hormonal therapies, but they also generally exhibit more-aggressive biologic behaviors and poorer prognosis than the receptor-positive ones. The results of the studies reported here show for the first time that the progesterone metabolites, 5αP and 3αHP, act as hormones that regulate ER/PR-negative breast tumor formation, growth, and regression. The onset of the ER/PR-negative human breast cell tumors in mice was considerably accelerated, and the growth significantly stimulated, by just one or two applications of 5αP. In contrast, 3αHP retarded onset of tumor formation, suppressed tumor growth, and inhibited or regressed existing 5αP-induced tumors. When both hormones were administered simultaneously, the effects of one were abrogated by the effects of the other. The current in vivo demonstrations, from five separate experiments, of the opposing actions of 5αP and 3αHP on tumorigenesis and tumor growth extend the previous findings, which showed opposing in vitro cancer-regulatory actions of these progesterone metabolites on receptor-negative (MDA-MB-231, MCF-10A) as well as receptor-positive (MCF-7, T47D, ZR-75-1) breast cell lines.
The response of ER/PR-negative breast cells to the progesterone metabolites can be explained by the presence of specific high-affinity receptors for 5αP (5αPR) and 3αHP (3αHPR). The 5αPR and 3αHPR (which are associated with the plasma membranes of both ER/PR-positive and ER/PR-negative cells) are distinct from each other and from known ER, PR, androgen, and corticosteroid receptors, and lack affinity for other steroids, such as progesterone, estrogen, androgens, corticosteroids, and other progesterone metabolites. Levels of 5αPR are upregulated by 5αP itself and estradiol, and downregulated by 3αHP in both ER/PR-positive and -negative cells. The mechanisms of action resulting in the opposing effects of these two hormones appear to involve cell-signaling pathways associated with the plasma membrane receptors, as well as altered gene expression. Indications are that 5αP acts via the surface receptor-linked mitogen-activated protein kinase (MAPK; Erk1/2) pathway; 5αP significantly stimulates activation of Erk1/2, increases the Bcl-2/Bax expression ratio and actin depolymerization, and decreases expression of actin and adhesion plaque-associated vinculin, resulting in decreased apoptosis and increased mitosis and cell detachment. Conversely, 3αHP appears to suppress protein kinase C (PKC), phospholipase C (PLC), Ca mobilization (unpublished observations), and the Bcl-2/Bax expression ratio, and increases expression of the cell-cycle inhibitor p21, resulting in increased apoptosis and decreased proliferation and detachment of breast cell lines. In pituitary cells, 3αHP also has been shown to inhibit a plasma membrane-associated PKC, PLC, Ca cell-signaling pathway.
The results of the studies reported here not only show that 5αP and 3αHP have opposing effects on initiation and growth of ER/PR-negative human breast tumors, but also provide in vivo evidence of the marked changes in the relative concentrations of these hormones in the tumor microenvironment. Whereas serum from control mice, in which implanted human breast cells had not developed into tumors, contained about equal concentrations of 5αP and 3αHP, serum from mice with tumors had significantly more 5αP than 3αHP. Because hormones had not been administered to these mice, the higher 5αP/3αHP ratio in serum from tumor-bearing mice can reasonably be expected to have resulted from the tumors, which on average had about threefold higher concentrations of 5αP than the respective sera, and >10-fold higher 5αP than 3αHP levels. Previous in vitro metabolism studies showed that human breast tumor tissues convert significantly more progesterone to 5α-pregnanes like 5αP and less to 4-pregnenes like 3αHP than do paired normal (nontumorous) tissues and that these differences correlated with significantly higher 5α-reductase gene (SRD5A1, SRD5A2) and lower 3α(20α)-HSO gene (AKR1C1, AKR1C2, AKR1C3) expression in tumor tissues. Similar differences in progesterone metabolism and enzyme gene expressions were observed between tumorigenic and nontumorigenic breast cell lines. In addition to the ability to convert progesterone to active cancer-regulating hormones, breast carcinomas are able to synthesize progesterone, which could account for its relatively high concentrations in the xenograft tumors reported here, and indicate an in situ supply of the biosynthetic precursor of 5αP and 3αHP.
The significant concentrations of 5αP and 3αHP, and particularly the high 5αP/3αHP ratios, in the MDA-MB-231 xenograft tumors, emphasize the potential importance of the microenvironment within breast tissue where the biologic actions occur. The role of the microenvironment in changing the expression of regulatory factors such as metabolizing enzymes, receptors, cytoskeletal and adhesion molecules, and growth promoters/inhibitors and in epigenetic alterations has been extensively reviewed. The current findings, along with the previous in vitro studies, suggest that the relative concentrations of 5αP and 3αHP in the breast microenvironment constitute important autocrine/paracrine determinants not only for tumorigenesis but also for potential regression of tumors and the maintenance of normalcy of ER/PR-negative breast cells/tissues. Figure 8 provides a summary of opposing biologic actions and proposed mechanisms of action of the progesterone metabolites, 5αP and 3αHP, in promoting neoplasia and tumorigenesis, as well as in maintaining normalcy in ER/PR-negative human breast cells. Evidence presented here shows that a high concentration of 5αP, relative to 3αHP in the microenvironment, promotes initiation and growth of tumors, whereas a higher concentration of 3αHP, relative to 5αP, suppresses tumorigenesis and promotes normalcy. Previous evidence indicates that these opposing effects of 5αP and 3αHP are propagated via the opposing actions of the hormones on cell proliferation (mitosis, apoptosis), adhesion, cell cycle, regulatory and signaling molecules, and gene expression after binding to specific receptors.
(Enlarge Image)
Figure 8.
Summary of the opposing autocrine/paracrine effects of the progesterone metabolites, 5αP and 3αHP, in a stylized ER/PR-negative human breast cell. Evidence presented here shows that a high concentration of 5αP relative to 3αHP, in the microenvironment, promotes initiation and growth of ER/PR-negative human breast cell tumors, whereas a higher concentration of 3αHP, relative to 5αP, suppresses tumorigenesis and promotes normalcy. Progesterone is converted to 3αHP and 5αP in breast cells. Tumorigenic and tumor cells convert more progesterone to 5αP and less to 3αHP than do normal cells. The steroids, being lipophylic, are able to pass out of cells and result in a concentration buildup in the microenvironment. The result is a significant increase in the 5αP-to-3αHP concentration ratio in the microenvironment of tumorigenic cells and within tumorous tissues in comparison with normal (nontumorous) breasts. 3αHP and 5αP bind to specific receptors on the plasma membrane linked to signaling pathways involving PKC, phospholipase C, and Ca mobilization (3αHP) and MAPK/Erk1/2 (5αP) and to modulators of gene expression. The cancer-inhibiting actions of 3αHP result in decreased proliferation and detachment of cells, increased apoptosis, and suppression of tumor initiation and growth. The cancer-promoting actions of 5αP have the opposite effects and result in stimulation of tumorigenesis and tumor growth. The evidence suggests that high concentrations of 5αP relative to 3αHP in the microenvironment will promote progression toward neoplasia and tumorigenesis, whereas a low 5αP-to-3αHP concentration ratio favors maintenance of the normal state.
How might higher levels of either 5αP or 3αHP in the serum due to the steroid implants have, respectively, initiated/promoted or suppressed xenograft tumorigenesis? The 5αP and 3αHP treatments, consisting of suspensions placed subcutaneously in the nape of the neck, resulted in elevated serum levels of either hormone, which persisted for about 2 to 3 weeks after the last injection. Because the depots were not far removed from the site of the cell implants, lymph drainage may have resulted in significantly higher concentrations of each applied hormone in the immediate vicinity of the implanted human cells. Conceivably, in the 5αP-treated mice, the induced elevation of 5αP levels, relative to 3αHP, in the microenvironment of the human cell implants, could have exerted procancer actions that initiated tumorigenesis. Because 5α-reductase and 5αPR levels are upregulated by 5αP, the in situ production and paracrine/autocrine actions of locally elevated 5αP could then have autoenhanced hormone-receptor interaction and the resulting stimulation of tumor growth, as illustrated in Figure 8. In a like manner, in the 3αHP-treated mice, the elevated 3αHP levels, relative to 5αP, in the microenvironment could have opposed progression to xenograft neoplasia by its inherent anticancer actions and the suppression of 5αP synthesis and 5αPR expression. By extension, in an intact human breast, local changes in relative concentrations of 5αP and 3αHP (that is, changes in the 5αP/3αHP ratio) resulting from selective up- or downregulation of progesterone-metabolizing enzymes induced by microenvironmental triggers could determine ER/PR-negative breast cell progression to tumor initiation and growth or maintenance of normalcy. Because only small changes in enzyme activity/expression are needed to result in significant local concentration changes, either a slight elevation of 5α-reductase, or a reduction of 3α-HSO, in one or more cells could lead to an increase in the ratio of 5αP/3αHP in the immediate intra- and extracellular environment. Conversely, processes that result in higher levels of cancer-suppressing 3αHP (and consequently lower 5αP/3αHP ratios) could ensure maintenance of normalcy.
Because in vitro studies have shown that both ER- and/or PR-negative and -positive breast cells respond in a stimulatory and inhibitory fashion, respectively, to 5αP and 3αHP, and have 5αPR and 3αHPR, the present results also may have implications for the substantial numbers (20% to 40%) of ER/PR-positive patients who fail to respond to suppression of estrogen and/or progesterone levels or actions, as well as for those receptor-positive tumors that do respond to hormonal therapies. In this regard, the opposing actions of the progesterone metabolites also appear to exert some control over the estrogen-regulated effects on breast cancer by their ability to modulate ER numbers in ER-positive cells. Furthermore, because both tumorigenic and normal (nontumorigenic) breast cells respond to the opposing actions of 5αP and 3αHP, the relative concentrations of the progesterone metabolites in the microenvironment may also play a role in maintaining normalcy of breast tissues in general, regardless of ER/PR status.
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