**8. E-RAF and mammary cancer**

E-RAF could play a role in the progression of mammary cancer. E-RAF II is a very active transcription factor and this molecular form of E-RAF represents more than 75%of total E-RAF population representing both E-RAFI and E-RAF II. If one assumes that the benign to malignant transformation of the mammary cancer is associated with enhanced expression of E-RAFII, that should be reflected in immunofluorescent detection of E-RAF in frozen

Evolving Trends in Estrogen Receptor Biology 73

found to be effective only in the presence of 15-16nM progesterone. The corresponding effects produced in the presence of different concentrations of cholesterol remains to be seen. The postulate that the enhancement in E-RAF titer in the pregnant uterus is an indirect reflection of the progesterone production in the uterus during the first half of pregnancy takes into account the possibility that mitochondrial steroidogenesis in the uterine cell is under E-RAF control. As mentioned earlier, E-RAF may function as a cholesterol transporter to the mitochondrial steroidogenic site, eventually facilitating the conversion of cholesterol to pregnenolone and progesterone. The nuclear genes influenced by cholesterol-bound E-RAF could well be those the products of expression of which are constituents of the

(1) Nucleus (2) Genes (3) Endoplasmic reticulum (4) Mitochondria (5) (5A) and (5B) Nuclear pore complex proteins that bind free E-RAF, progesterone-bound E-RAF and cholesterol bound E-RAF respectively. (6) ap55 (6A) Estradiol-bound ap55 (7) tp66 (7A) tp66-E-RAF complex (8) Free E-RAF (9)

The primary site of intracellular location of E-RAF is the endoplasmic reticulum where it remains anchored to the anchor protein 55(ap55) through the mediation of tp66 (transport protein 66) in an estrogen dependent manner.tp66 transports E-RAF to the nucleus, after dissociation from ap55, when the intracellular level of estrogen declines. When bound by cholesterol or progesterone, E-RAF dissociates from tp66 and moves independently to the nucleus. The nuclear entry of E-RAF again is regulated by nuclear pore complex proteins (npcp). Apparently it is the conformation of E-RAF that determines the identity of npcp with which it should interact. There is a distinct possibility that within the nucleus free E-RAF, progesterone bound E-RAF and cholesterol bound E-RAF recognize

Progesterone bound E-RAF (10) Cholesterol bound E-RAF. Fig. 5. **Mode of action of E-RAF in the target cell.**

mitochondrial steroidogenic complex like cytochrome P450(Fig.5)

biopsies of mammary tissue. Figure 4 presents the results of a recent study carried out in this direction where the tissue sections were exposed first to anti E-RAF IgG and subsequently to FITC labeled secondary antibody. There is a dominating presence of E-RAF in the cytoplasm and also in the nuclei (primarily stained with propidium iodide).

A cryostat section of human breast cancer biopsy was fixed, permeabilised and exposed overnight to rabbit anti goat E-RAF IgG at 40C.Further exposure of the section to goat anti rabbit IgG labeled with FITC was conducted in the dark for 1 hr following which the nuclei were stained with propidium iodide. The sections were examined using a Leica fluorescence microscope. The green fluorescence indicates the cytoplasmic presence of E-RAF.The nuclei are stained red with propidium iodide. However the presence of E-RAF in the nuclei is marked by the transition of the red colour to light orange and even yellow.
