INTERACTION OF SONIC HEDGEHOG PROTEIN WITH ESTROGEN AND ANDROGEN RECEPTORS IN BREAST CARCINOMA

Cilj istraživanja : Aktivnost HH-GLI signalnog kao i receptora androgena opisana je u karcinomu dojke, no i dalje bez jasnog mehanizma djelovanja pa tako i jasne uloge. U ovom istraživanju na 186 arhivskih uzoraka karcinoma dojke smo ustanovili ekspresiju proteina SHH i receptora androgena te istražili povezanost tih ekspresija s kliničko-patološkim značajkama karcinoma dojke i pojedinih molekularnih podtipova. Istražili smo povezanost ekspresija navedenih proteina s ukupnim preživljenjem, preživljenjem do pojave recidiva, odnosno metastaza te pojavnošću smrti, recidiva i metastaza.Također smo ispitali mogućnost fizičke interakcije proteina SHH s receptorom androgena i estrogena potaknuti nedavnom spoznajom o neposrednoj fizičkoj interakciji proteina SHH i receptora androgena na staničnom modelu karcinoma prostate. Nacrt studije : Istraživanje je provedeno kao presječno s povijesnim podatcima. Materijali i metode : Korišten je arhivski materijal Kliničkog zavoda za patologiju KB Dubrava. Odabrani su uzorci karcinoma dojke žena operiranih u periodu 2010.g.-2015.g. koji su konsekutivno razvrstavani u skupine prema molekularnom podtipu do planiranog broja uzoraka za svaku podskupinu. Imunohistokemijski su se odredile ekspresije proteina SHH i receptora androgena, a fizička interakcija istražila se pomoću PLA (proximity ligation assay) metode na staničnim linijama MCF-7 i SkBr3. Rezultati : Nije pronađena statistički značajna povezanost između ekspresije proteina SHH i kliničko-patoloških značajki te ishoda iako su veće vrijednosti ekspresije u hormonski pozitivnih tumora, luminalnih podtipova, tumora s manje zahvaćenih limfnih čvorova isto tako veća ekspresija SHH je povezana s manje učestalim smrtnim ishodom, razvojem recidiva i metastaza. Visoka ekspresija proteina SHH značajno je povezana s ranijim razvojem recidiva (P=0,022) iako je uočena na malenom broju uzoraka. Ukupno preživljenje kraće je u bolesnika s većom ekspresijom proteina SHH, no bez statističke značajnosti kao i kasniji razvoj metastaza. Ustanovljena je značajna povezanost visoke ekspresije AR i prisutnosti receptora estrogena i progesterona, odnosno s luminalnim podtipovima (P < 0,001), karcinoma s in-situ komponentom (P = 0,01), karcinoma s Ki67 < 20 % (P = 0,0001) te manjim karcinomima (P = 0,007). PLA metodom u našem istraživanju nije dokazana fizička interakcija proteina SHH s receptorima estrogena i androgena. Prema opažanjima u ovom istraživanju visoke ekspresije recptora androgena i proteina SHH povezane s povoljnijim kliničko-patološkim karakteristikama i ishodom.Objectives: The activation of HH-GLI signalling pathway as well as the androgen receptor have been described in breast cancer, but a clear pathophysiological mechanism of action and thus a clear role still remains unknown. In this study, we determined the expression of the SHH protein and the androgen receptor on 186 archival samples of breast cancer and researched the correlation of these expressions with clinical and pathological features of breast cancer and individual molecular subtypes. We researched the correlation of the expression of the above-mentioned proteins with overall survival, recurrence free survival, metastasis free survival, and the occurrence of death, recurrence, and metastases. Prompted by the recent knowledge of the direct physical interaction of the SHH protein and the androgen receptor on prostate cancer cell lines, we also examined the possibility of physical interaction of the SHH protein with the androgen and estrogen receptors. Study design: Cross-sectional study with archival data. Materials and methods: Studied samples were obtained from the Pathology Department archive, Clinical Hospital Dubrava. Breast cancer samples were selected from women operated on in the period of 2010-2015. Samples were consecutively classified into groups according to molecular subtype up to the planned number of samples for each subgroup. Expressions of SHH protein and androgen receptor were determined immunohistochemically, and physical interaction was researched using the PLA (proximity ligation assay) method on MCF-7 and SkBr3 cell lines. Results: No statistically significant association was found between SHH protein expression and clinical-pathological features and outcomes, although higher expression values were present in hormone-positive tumors, luminal subtypes, tumors with fewer affected lymph nodes. Higher SHH expression is also associated with less frequent death, relapse, and metastasis. There was a statistically significant association between high SHH protein expression and earlier development of recurrence (P = 0.022), although it was observed in a small number of samples. Overall survival is shorter for patients with higher SHH protein expression, but without a statistical significance, as well as the later development of metastases. A significant association was found between high AR expression and the presence of estrogen and progesterone receptors, i.e., with luminal subtypes (P < 0.001), carcinoma with 'in-situ' component (P = 0.01), carcinoma with Ki67 < 20% (P = 0.0001) and smaller cancers (P = 0.007). Physical interaction of SHH protein with estrogen and androgen receptors was not proven in our research using the PLA method. Findings of this study showed that high expressions of SHH protein and androgen receptor are associated with favourable clinical and pathological features and a more favourable outcome