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Heregulin (HRG) and type I receptor tyrosine kinase (RTK) expression was investigated in the highly invasive and metastatic LM3 cell line, our previously described model of metastasis for mammary cancer (Bal de Kier Joffe et al. [1986] Invasion Metastasis 6:302-12; Urtreger et al. [1997] Int J Oncol 11:489-96). Although LM3 cells do not express HRG, they exhibit high levels of ErbB-2 and ErbB-3 as well as moderate expression of ErbB-4. Addition of exogenous HRGbeta1 resulted in inhibition of both proliferation and migration of LM3 cells. HRGbeta1 was also able to decrease the activity of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9), 2 key enzymes in the invasion and metastatic cascade. HRGbeta1 treatment of LM3 cells induced tyrosine phosphorylation of ErbB-2, ErbB-3 and ErbB-4 as well as the formation of ErbB-2/ErbB-3 and ErbB-2/ErbB-4 heterodimers. Assessment of the signaling pathways involved in HRGbeta1 action indicated that the addition of HRGbeta1 to LM3 cells resulted in activation of phosphatidylinositol 3- kinase (PI-3K) and in strong induction of the association of the p85 subunit of PI-3K with ErbB-3. HRGbeta1 also caused the rapid activation of ERK1/ERK2 and Stat3 and Stat5 (signal transducers and activators of transcription [STAT]). This is the first demonstration of the ability of HRGbeta1 to activate STATs in mammary tumor cells. Blockage of PI-3K activity with its chemical inhibitor wortmannin, or of MEK1/ERKs activity with PD98059, resulted in suppression of the ability of HRGbeta1 to inhibit LM3 cell growth. Notwithstanding the suppression of these 2 signaling pathways, HRGbeta1 still proved capable of inhibiting uPA activity. Therefore, our results provide evidence that signaling pathways involved in HRGbeta1-induced proliferation appear to be distinct from those involved in HRGbeta1 regulation of uPA, a protease that plays a pivotal role in invasion and metastasis., (Copyright 2002 Wiley-Liss, Inc.)

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.

To evaluate the possible involvement of the salivary glands in the modulation of medroxyprogesterone (MPA)-induced mammary tumorigenesis, 48 sialoadenectomized virgin BALB/c female mice and 47 controls were treated with 40mg MPA depot s.c. every 3 months for 1 year. Mammary tumors developed in 11 sialoadenectomized and in 34 control mice with similar latencies. In both groups, 75% of the tumors were ductal and progestin-dependent (PD) while the remainder were lobular and progestin-independent (PI). Epidermal growth factor (EGF) levels were measured in salivary glands (SG-EGF) and serum (S-EGF) in both groups. MPA induced a significant increase in SG-EGF and in S-EGF that became evident only after 1 month of MPA treatment. No increase in S-EGF was detected in MPA-treated sialoadenectomized mice, indicating that salivary glands are the major source of S-EGF. The presence of EGF receptors (EGF-R) was investigated in ductal PD and PI tumor lines and compared with 8 PI tumor lines of lobular origin. A significant difference in EGF-R content was found between lobular and ductal tumors. No increase in EGF-R was noted when ductal tumors became autonomous. EGF-R did not correlate with tumor growth rate and there was an inverse correlation between EGF-R and steroid receptors. When the effect of sialoadenectomy on tumor growth was tested in vivo in syngeneic transplants of 2 ductal PD, 1 ductal PI and 2 lobular PI mammary adenocarcinomas, it was not found to be significant when compared with the controls. It may be concluded that SG-EGF plays an important role in the induction of mammary adenocarcinomas by MPA, while it has no significant effect on the growth of established tumors.

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.

The interaction of estradiol-receptor complexes with isolated human breast tumor chromatin was studied under equilibrium conditions. Tumor samples were obtained from mastectomy specimens or biopsies. Chromatin was prepared from these samples and incubated with estradiolreceptor partially purified from cytosol rat uterus or from human hormone-dependent tumors. The two preparations gave similar results in the interactions with human chromatin. The in vitro transcription activity of chromatin from hormone-dependent tumors was dramatically increased by the addition of estradiol-receptor. Maximal enhancement was achieved with 70 fmoles of estradiol-receptor complex in the presence of 50 micrograms of chromatin. These changes in RNA synthesis were not detectable with chromatin isolated from autonomous breast tumors.

Mammary adenocarcinomas were induced by medroxy-progesterone acetate (MPA) in female BALB/c mice. From 5 primary tumors, 9 different sublines were established by s.c. transplantation into syngeneic female mice; these developed after a long latent period (4-12 months). Each subline was transplanted both into 4 mice treated with 40mg of MPA depot (s.c. contralaterally to the tumor inoculum) and into 4 non-treated mice. Of the 9 sublines, 6 proved to be hormone-dependent (MPA-D) and 3 hormone-independent or autonomous (MPA-I). However, even the autonomous lines, when treated with MPA, showed a slight increase in growth. All MPA-D lines had a high content of ER (20-254 fmoles/mg of protein), PR (63-710), PRL-R (44-74) and low or non-detectable EGF-R. Of the 3 MPA-I sublines that were studied, 2 showed a high content of ER (16-125), PR (27-708), PRL-R (19-70) and EGF-R (29-65) while the other one had a low content of ER (0-36), PR (0-13), no EGF-R and moderate PRL-R (15-52). Spontaneous mammary tumors of BALB/c and C3H origin, which also showed an MPA-I pattern of tumor growth, had high levels of EGF-R. We postulate that MPA has a direct effect on mammary tumor cells in MPA-D lines and that the expression of EGF-R is correlated with an autonomous pattern of growth.

Analysis of estrogen and progesterone cytosolic receptors (ER, PR) has been studied in human breast carcinoma. ER were assayed by sucrose density gradients and dextran coated-charcoal method. In a series of 109 ER+ breast tumors, 64.2% exhibited the 8S form, 25% the 4S and 11% both the 8S and 4S components. From a total number of 970 specimens, 776 (80%) were primary and 194 (20%) metastatic lesions. In the whole group, ER was positive in 77.6% and PR in 61.4%, while both receptors were found simultaneously in 59%. The concentration of ER was higher in postmenopausal women in comparison to premenopausal women. On the other hand, the levels of PR were rather similar in both groups. In primary lesions, PR was more frequently seen than in secondary tumors and the same holds true for the presence of ER and PR and the frequency of negative tumors. Specific prolactin (PRL) binding assay was performed in a group of 50 breast tumors. Positive and negative distribution was almost equal (48 vs 52%) and there was not correlation between PRL-R and ER. In addition, a limited group of male breast cancer, showed that three out of five patients possess only androgen receptors, whereas the remaining two cases were exclusively ER+. In summary, the present results indicate that the local incidence of steroid receptors in female breast carcinoma, are very likely similar to previous reports either in premenopausal and postmenopausal women and in primary and secondary lesions. The importance of PRL-R assay requires a further evaluation.