MiR-16 mediates trastuzumab and lapatinib response in ErbB-2-positive breast and gastric cancer via its novel targets CCNJ and FUBP1.

ErbB-2 amplification/overexpression accounts for an aggressive breast cancer (BC) subtype (ErbB-2-positive). Enhanced ErbB-2 expression was also found in gastric cancer (GC) and has been correlated with poor clinical outcome. The ErbB-2-targeted therapies trastuzumab (TZ), a monoclonal antibody, and lapatinib, a tyrosine kinase inhibitor, have proved highly beneficial. However, resistance to such therapies remains a major clinical challenge. We here revealed a novel mechanism underlying the antiproliferative effects of both agents in ErbB-2-positive BC and GC. TZ and lapatinib ability to block extracellular signal-regulated kinases 1/2 and phosphatidylinositol-3 kinase (PI3K)/AKT in sensitive cells inhibits c-Myc activation, which results in upregulation of miR-16. Forced expression of miR-16 inhibited in vitro proliferation in BC and GC cells, both sensitive and resistant to TZ and lapatinib, as well as in a preclinical BC model resistant to these agents. This reveals miR-16 role as tumor suppressor in ErbB-2-positive BC and GC. Using genome-wide expression studies and miRNA target prediction algorithms, we identified cyclin J and far upstream element-binding protein 1 (FUBP1) as novel miR-16 targets, which mediate miR-16 antiproliferative effects. Supporting the clinical relevance of our results, we found that high levels of miR-16 and low or null FUBP1 expression correlate with TZ response in ErbB-2-positive primary BCs. These findings highlight a potential role of miR-16 and FUBP1 as biomarkers of sensitivity to TZ therapy. Furthermore, we revealed miR-16 as an innovative therapeutic agent for TZ- and lapatinib-resistant ErbB-2-positive BC and GC.

Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.

Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance.

Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a critical role in breast cancer (BC). We and others have also shown the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. Current anti-ErbB-2 therapies, as with the antibody trastuzumab (Ttzm), target only MErbB-2. Here, we found that blockade of NErbB-2 action abrogates growth of BC cells, sensitive and resistant to Ttzm, in a scenario in which ErbB-2, ErbB-3 and Akt are phosphorylated, and ErbB-2/ErbB-3 dimers are formed. Also, inhibition of NErbB-2 presence suppresses growth of a preclinical BC model resistant to Ttzm. We showed that at the cyclin D1 promoter, ErbB-2 assembles a transcriptional complex with Stat3 (signal transducer and activator of transcription 3) and ErbB-3, another member of the ErbB family, which reveals the first nuclear function of ErbB-2/ErbB-3 dimer. We identified NErbB-2 as the major proliferation driver in Ttzm-resistant BC, and demonstrated that Ttzm inability to disrupt the Stat3/ErbB-2/ErbB-3 complex underlies its failure to inhibit growth. Furthermore, our results in the clinic revealed that nuclear interaction between ErbB-2 and Stat3 correlates with poor overall survival in primary breast tumors. Our findings challenge the paradigm of anti-ErbB-2 drug design and highlight NErbB-2 as a novel target to overcome Ttzm resistance.

Progestin-induced caveolin-1 expression mediates breast cancer cell proliferation.

Progestin regulation of gene expression was assessed in the progestin-dependent murine tumor line C4HD which requires MPA, a synthetic progestin, for in vivo growth and expresses high levels of progesterone receptor (PR). By using suppressive subtractive hybridization, caveolin-1 was identified as a gene whose expression was increased with in vivo MPA treatment. By Northern and Western blot analysis, we further confirmed that caveolin-1 mRNA and protein expression increased in MPA-treated tumors as compared with untreated tumors. When primary cultures of C4HD cells were treated in vitro with MPA, caveolin-1 levels also increased, effect that was abolished by pre-treatment with progestin antagonist RU486. In addition, MPA promoted strong caveolin-1 promoter transcriptional activation both in mouse and human breast cancer cells. We also showed that MPA regulation of caveolin-1 expression involved in activation of two signaling pathways: MAPK and PI-3K. Short-term MPA treatment of C4HD cells led to tyrosine phosphorylation of caveolin-1 protein, where Src was the kinase involved. Additionally, we showed that MPA-induced association of caveolin-1 and PR, which was detected by coimmunoprecipitation and by confocal microscopy. Finally, we proved that MPA-induced proliferation of C4HD cells was inhibited by suppression of caveolin-1 expression with antisense oligodeoxynucleotides to caveolin-1 mRNA. Furthermore, we observed that inhibition of caveolin-1 expression abrogated PR capacity to induced luciferase activity from a progesterone response element-driven reporter plasmid. Comprehensively, our results demonstrated for the first time that caveolin-1 expression is upregulated by progestin in breast cancer. We also demonstrated that caveolin-1 is a downstream effector of MPA that is partially responsible for the stimulation of growth of breast cancer cells.

Bloqueo del receptor del factor de crecimiento semejante a la insulina tipo I utilizando oligodeoxinucleótidos antisentido en cáncer de mama experimental / Type I insulin-like growth factor receptor antisense strategies in experimental breast cancer

We addressed the effect of targeting type I insulin-like growth factor receptor (IGF-IR), with antisense strategies in in vivo growth of breast cancer cells. We used C4HD tumors from an experimental model of hormonal carcinogenesis in which medroxyprogesterone acetate induced mammary adenocarcinomas in Balb/c mice. Intratumor or systemic administration of phosphorothiolated antisense oligodeoxynucleotides (AS[S]ODN) to IGF-IR mRNA resulted in a significant inhibition of C4HD tumor growth. The antitumor effect was specific since inhibition of tumor growth was dose-dependent and no effect was observed in mice treated with sense S[S]ODN. Tumors from AS[S]ODN-treated mice showed a decrease in IGF-IR expression and in insulin receptor substrate-1 tyrosine phosphorylation. Activation of PI-3K/Akt, p42/p44 MAPK and ErbB-2 was abolished in tumors treated with AS[S]ODN. Progesterone receptor expression or activity remained invariable. This is the first demonstration that breast cancer growth can be inhibited by direct in vivo administration of IGF-IR AS[S]ODN.

Evaluamos el efecto del bloqueo de la expresión del receptor del factor de crecimiento semejante a lainsulina tipo I (IGF-IR) sobre el crecimiento in vivo de cáncer de mama empleando una estrategia“antisentido”. Utilizamos el adenocarcinoma mamario murino progestágeno-dependiente C4HD. La administración intratumoral o sistémica de oligodeoxinucleótidos antisentido fosfotiolados al ARNm del IGF-IR (AS[S]ODN) inhibió el crecimiento tumoral. El efecto antitumoral fue específico debido a su dosis-dependencia y a la faltade efecto en ratones tratados con el S[S]ODN “sentido”. Los tumores obtenidos de ratones tratados con AS[S]ODN mostraron: disminución en la expresión de IGF-IR y en la fosforilación del sustrato del receptor de insulina-1, inhibición de la activación de PI-3K/Akt, p42/p44MAPK y ErbB-2, mientras que la expresión y activación del receptor de progesterona no se afectó. Es la primera demostración que el

Mechanisms of cell cycle arrest in response to TGF-beta in progestin-dependent and -independent growth of mammary tumors.

TGF-beta1 modulation of cell cycle components was assessed in an experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary tumors in Balb/c mice. TGF-beta1 inhibited both MPA-induced proliferation of progestin-dependent C4HD epithelial cells and proliferation of the progestin-independent variant cell type C4HI, arresting cells in G(1) phase of the cell cycle. Progestin-independent 60 epithelial cells evidenced reduced response to TGF-beta1 antiproliferative effects. TGF-beta1 inhibition of cyclins D1 and A expression and up-regulation of p21(CIP1) levels were the common findings in all three cell types. In addition, a significant content reduction of cyclin D1/cdk4 and cyclin A/cdk2 complexes was found after TGF-beta1 inhibition of MPA-dependent and -independent proliferation. TGF-beta1 inhibited cyclin D2 expression and up-regulated p27(KIP1) levels only when acting as inhibitor of MPA-induced proliferation of C4HD cells. Regulation of these two cell cycle components resulted in decreased cyclin D2/cdk2 complex and in increased p27(KIP1) association with cdk2 in C4HD cells treated with TGF-beta1. These two molecular mechanisms, unobserved in progestin-independent growth of C4HI or 60 cells, were associated with a significantly higher degree of inhibition of cdk2 kinase activity in C4HD cells compared to that found in TGF-beta-treated C4HI or 60 cells. Reduced sensitivity of 60 cells to the growth-inhibitory effects of TGF-beta1 correlated with significantly lower levels of p15(INK4B), p21(CIP1), and p27(KIP1) expressed in these cells, compared to the levels present in C4HD or C4HI cells, and correlated as well with lack of expression of p16(INK4). Thus, common targets were found to exist in TGF-beta1 inhibitory action on breast cancer cells, but regulation of specific targets was found when TGF-beta1-inhibited proliferation driven by the progesterone receptor.

Activation of ErbB-2 via a hierarchical interaction between ErbB-2 and type I insulin-like growth factor receptor in mammary tumor cells.

The present study focused on interactions between signaling pathways activated by progestins and by type I and II receptor tyrosine kinases (RTKs) in mammary tumors. An experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice was used. MPA-stimulated proliferation, both in vivo and in vitro, of progestin-dependent tumors induced up-regulation of ErbB-2 protein levels and tyrosine phosphorylation of this receptor. Combinations of antisense oligodeoxynucleotides (ASODNs) directed to ErbB-2 mRNA with ASODNs directed to the insulin-like growth factor-I receptor (IGF-IR) were used to study the effect of the simultaneous block of these receptors on the MPA-induced proliferation of epithelial cells from the progestin-dependent C4HD line. Neither synergistic nor additive effects on the inhibition of MPA-induced proliferation of C4HD cells were observed as a result of the combination of these ASODNs. Suppression of IGF-IR expression by ASODNs resulted in complete abrogation of MPA-induced phosphorylation of ErbB-2 in C4HD cells, whereas blockage of ErbB-2 did not affect IGF-IR phosphorylation. These results show the existence of a hierarchical interaction between IGF-IR and ErbB-2, by means of which IGF-IR directs ErbB-2 phosphorylation. We demonstrated, for the first time, that this hierarchical interaction involves physical association of both receptors, resulting in the formation of a heteromeric complex. Furthermore, confocal laser microscopy experiments demonstrated that MPA was able to induce co-localization of ErbB-2 and IGF-IR. This hetero-oligomer was also found in MCF-7 human breast cancer cells in which association of IGF-IR and ErbB-2 was induced by heregulin and IGF-I. Oncogene (2001) 20, 34 - 47.

Effects of antiprogestins RU486 and ZK98299 on the expression of cell cycle proteins of a medroxyprogesterone acetate (MPA)-induced murine mammary tumor.

This study links the tumor inhibitory effect of anti-progestins RU486 and ZK98299 to the expression of cell cycle proteins. Medroxyprogesterone acetate-induced ductal mammary adenocarcinoma-bearing female BALB/c mice were treated daily either with RU486 or ZK98299, observing tumor growth retardation. p21 increased after 24 h treatment, peaked at day 4 and returned to control levels at day 7. Cyclin D1, cyclin E, CDK2 and CDK4, did not change during treatment. These results suggest that p21 might play a role in early inhibitory stages of tumor growth induced by RU486 and ZK98299.

Interactions between progestins and heregulin (HRG) signaling pathways: HRG acts as mediator of progestins proliferative effects in mouse mammary adenocarcinomas.

The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.

Differential expression of and responsiveness to transforming growth factor-beta (TGF-beta) isoforms in hormone-dependent and independent lines of mouse mammary tumors.

Transforming growth factor-beta2 (TGF-beta2) and -beta3 mRNA expressions were studied in ductal hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. MPA treatment of HD tumors induced a significant decrease in TGF-beta2 and -beta3 mRNA levels. Progression to an HI phenotype of ductal tumors was associated with reduced TGF-beta2 and -beta3 expressions, as compared with their HD counterparts. Exogenously added TGF-beta1, -beta2, and -beta3 (1 ng/ml) inhibited the proliferation of primary cultures of epithelial cells from ductal HD and HI tumors. In addition, TGF-beta expression and effects were studied in the other type of MPA-induced mammary tumors, which are of lobular origin and lack steroid hormone receptors and evidence an HI behavior. These lobular HI lines showed TGF-beta2 levels similar to those found in HD lines growing in MPA-treated mice. In contrast, TGF-beta3 mRNA levels were 12- to 20-fold higher than in HD tumors. Primary cultures of lobular HI epithelial cells required either TGF-beta concentrations of 10 ng/ml to show an inhibitory response, or were completely resistant to TGF-beta inhibition. Studies of the molecular mechanisms involved in reduction or loss of TGF-beta responsiveness in lobular HI tumors showed that cell surface type II TGF-beta receptor levels were lower in these tumors than those present in HD tumors. Our results support the hypothesis that TGF-beta could play a role as an autocrine growth inhibitor in HD and HI ductal tumors. Autonomous growth of lobular HI tumors could be favored by undetectable or low TGF-beta1 and -beta2 expressions and by reduced or lost sensitivity of epithelial cells to TGF-beta's antiproliferative effects. However, the extremely high levels of TGF-beta3 expression in lobular HI tumors, in spite of reduced sensitivity to TGF-beta3 inhibitory growth effect in tumor epithelial cells, suggest a net positive role for TGF-beta3 in these tumors.

Involvement of insulin-like growth factors-I and -II and their receptors in medroxyprogesterone acetate-induced growth of mouse mammary adenocarcinomas.

The role of the insulin-like growth factors (IGFs) system was investigated in hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. IGF-II protein and message showed a three- to four-fold increase in HD lines growing in MPA-treated mice, as compared with HD tumors growing in untreated mice. Progression to a hormone-independent phenotype in all these lines was accompanied by a high constitutive expression of IGF-II. Similar IGF-I mRNA levels were detected in HD and HI lines. Both IGF-I and -II messages arose from the malignant epithelial cells, as shown by in situ hybridization studies. A significant decrease in Man-6P/type II IGF-R content was detected in HD tumors growing in MPA-treated mice as compared with HD lines growing in untreated mice. On the other hand, in HI tumors, notwithstanding high IGF-II synthesis, the levels of Man-6P/type II IGF-R remain high. Competitive inhibition and affinity labeling studies showed an almost exclusive binding of IGF-II to Man-6P/type II IGF-R on tumor membranes. The involvement of IGFs in the growth of epithelial primary cultures of the C4-HD line was evaluated. Exogenous IGF-I potentiated MPA stimulatory effect at concentrations of 50-100 ng/ml. Treatment of C4-HD cells with antisense oligodeoxynucleotides (ASODNs) to type I IGF-R and to IGF-II RNA resulted in a dose-dependent inhibition of MPA-mediated cell proliferation. The inhibition caused by IGF-II ASODNs could not be overcome by the addition of IGF-II up to 150 ng/ml. ASODNs to type I IGF-R at 40 microg/ml reduced by 75% the number of type I IGF-R; ASODNs to IGF-II at 1 microM decreased by 83% the levels of IGF-II protein. Our results provide support for the involvement of IGF-I and -II in MPA-induced mammary tumor growth by autocrine pathways.

[Growth hormones and oncogenes in mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice]. / Factores de crecimiento y oncogenes en adenocarcinomas mamarios inducidos por acetato de medroxyprogesterona en ratones BALB/c.

We have studied the involvement of growth factors (GF), their receptors (GF-R) and oncogenes in modulating tumor growth in the medroxyprogesterone acetate (MPA)-induced mammary tumor model in BALB/c mice. We demonstrated the presence of both ligands of the insulin-like growth factor family (IGF-I, IGF-II) and the two types of receptors (IGF-RI, IGF-RII). MPA upregulated IGF-II mRNA and protein levels in hormone-dependent lines (MPA-D). The progression to a hormone-independent phenotype was accompanied by a high constitutive expression of IGF-II and by a significant decrease in IGF-IIR number. An antisense strategy used to evaluate the role of IGF in the MPA-induced growth of epithelial MPA-D cells showed that IGF mediate progestin-induced mammary tumor growth by autocrine/intracrine pathways. We also studied the role of heregulins (HRG), the recently identified ligands for the c-erbB3 and c-erbB4 oncogenes. HRG mRNA expression was restricted to tumors of ductal origin. MPA induced an in vivo up-regulation of HRG expression. Finally, we also found that MPA may be exerting its proliferative effect on MPA-D lines by inhibiting the expression of transforming growth factor beta 1, (TGF-beta 1) and the lack of expression of TGF-beta 1 in hormone-independent tumors may be related to the acquisition of autonomous growth.

Varying patterns of expression of insulin-like growth factors I and II and their receptors in murine mammary adenocarcinomas of different metastasizing ability.

We studied the expression of insulin-like growth factors I (IGF-I) and II (IGF-II) and their receptors (IGF-R) in 2 related murine mammary adenocarcinoma in vivo lines, M3 and MM3, with different metastasizing ability. We further investigated the effects of IGFs on the secretion of a key enzyme in the metastatic cascade, the urokinase-type plasminogen activator (uPA) in M3 and MM3 cells. M3 is a spontaneous mammary tumor originated in BALB/c mice, with a 40% incidence of lung metastases. MM3 variant, obtained by successive s.c. implants of M3 lung metastases into syngeneic mice, shows a 95% incidence of lung metastases. Similar levels of expression of IGF-I protein were found in M3 and MM3 tumors, whereas IGF-II expression was 4-fold higher im MM3. RNAse protection assays showed similar levels of IGF-I mRNA in M3 and MM3 tumors and revealed a 4-fold increase in IGF-II transcripts in MM3 tumors compared with M3. Authentic IGF-I and II messages were also found in primary cultures of M3 and MM3 cells. IGF-I mRNA levels were similar in both cultures and, as described for solid tumors a 5-fold increase in IGF-II message was detected in MM3 cells. The presence of type I and mannose-6-phosphate (Man-6P)/type II IGF-R was demonstrated in both M3 and MM3 tumors. A 2-fold increase of type I IGF-R was detected in MM3 tumors compared with M3. Man-6P/type II IGF-R levels were 2-fold lower in MM3 tumors than in M3. As observed in tumor membranes, type I IGF-R concentrations were higher and Man-6P/type II IGF-R lower in cultures of MM3 epithelial cells compared with MM3 cells. In addition, we found that IGF-I enhanced secreted uPA activity in both M3 and MM3 cells while IGF-II only stimulated uPA secretion in MM3 cells.

Correlation of TGF-beta 1 expression with medroxyprogesterone acetate responsiveness in mouse mammary adenocarcinomas.

We investigated the expression of transforming growth factors beta 1 and alpha (TGF-beta 1, TGF-alpha) in hormone-responsive (MPA-R) and unresponsive (MPA-U) tumor lines obtained from medroxyprogesterone acetate (MPA)-induced mammary adenocarcinomas in BALB/c mice. The tumors were transplanted into MPA-treated and untreated mice. TGF-beta 1 gene expression was observed in the MPA-R lines growing in untreated animals, but not in MPA-treated mice. TGF-beta 1 mRNA was not detected in the MPA-U tumor lines growing in either MPA-treated or untreated animals. In MPA-R lines the levels of TGF-beta 1 expression were inversely correlated to growth rate. High-affinity TGF-beta 1 receptors were present in the MPA-R tumors. These results suggest that one of the mechanisms by which MPA exerts its proliferative effect on MPA-R tumor lines is inhibition of the expression of TGF-beta 1. Thus, the lack of expression of TGF-beta 1 in MPA-U tumors may be related to the acquisition of autonomous growth.

Enhanced expression of angiotensin II receptor subtypes and angiotensin converting enzyme in medroxyprogesterone-induced mouse mammary adenocarcinomas.

Angiotensin II receptors of the AT1 subtype were very highly expressed in medroxyprogesterone-induced ductal adenocarcinomas of the mammary gland in BALB/c mice. AT1 receptors are associated only to neoplastic epithelial cells. Lobular adenocarcinomas expressed very few AT1 receptors and expressed AT2 receptors only in areas corresponding to peritumoral connective tissue. Binding to angiotensin converting enzyme was present in all adenocarcinomas studied and was higher in ductal than in lobular adenocarcinomas. Normal mammary gland did not express either angiotensin II receptors or angiotensin converting enzyme. The present results are the first demonstration of angiotensin receptor subtypes and converting enzyme in mammary adenocarcinomas differentially expressed in tumors of ductal and lobular origin. Localization of receptor subtypes could be useful to study the differentiation of mammary cells during experimental mammary carcinogenesis in mice.

Estrogen inhibition of MPA-induced mouse mammary tumor transplants.

Estrogen compounds were used to treat mice bearing syngeneic transplants of medroxyprogesterone acetate(MPA)-induced BALB/c mammary adenocarcinomas. Both MPA-dependent and MPA-independent tumor lines were used. These lines expressed estrogen (ER) and progesterone receptors (PR). We demonstrate that different doses of estradiol benzoate (EB) and 17-beta-estradiol (E2) inhibit tumor growth and induce tumor regression in both MPA-independent and -dependent tumors, even in the presence of MPA or progesterone (P). EB was unable to induce regression of (ER-) hormone-independent tumor lines. A few MPA-dependent tumors became resistant to the estrogenic treatment; in subsequent passages some of these tumors retained their MPA-responsiveness, although estrogen sensitivity was not recovered.

Transforming growth factor-beta activities in 'in vivo' lines of hormone-dependent and independent mammary adenocarcinomas induced by medroxyprogesterone acetate in BALB/c mice.

We have determined the presence of transforming growth factor-beta (TGF-beta)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in 'in vivo' HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-beta from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-beta antibody.