Therapeutic effects of deleting cancer-associated fibroblasts in cholangiocarcinoma

Abstract

Cancer-associated fibroblasts (CAF) are abundant in the stroma of desmoplastic cancers where they promote tumor progression. CAFs are "activated" and as such may be uniquely susceptible to apoptosis. Using cholangiocarcinoma as a desmoplastic tumor model, we investigated the sensitivity of liver CAFs to the cytotoxic drug navitoclax, a BH3 mimetic. Navitoclax induced apoptosis in CAF and in myofibroblastic human hepatic stellate cells but lacked similar effects in quiescent fibroblasts or cholangiocarcinoma cells. Unlike cholangiocarcinoma cells, neither CAF nor quiescent fibroblasts expressed Mcl-1, a known resistance factor for navitoclax cytotoxicity. Explaining this paradox, we found that mitochondria isolated from CAFs or cells treated with navitoclax both released the apoptogenic factors Smac and cytochrome c, suggesting that they are primed for cell death. Such death priming in CAFs appeared to be due, in part, to upregulation of the proapoptotic protein Bax. Short hairpin RNA-mediated attenuation of Bax repressed navitoclax-mediated mitochondrial dysfunction, release of apoptogenic factors, and apoptotic cell death. In a syngeneic rat model of cholangiocarcinoma, navitoclax treatment triggered CAF apoptosis, diminishing expression of the desmoplastic extracellular matrix protein tenascin C, suppressing tumor outgrowth, and improving host survival. Together, our findings argue that navitoclax may be useful for destroying CAFs in the tumor microenvironment as a general strategy to attack solid tumors.

Figure 1. Navitoclax induces apoptosis in human CAF, but not CCA cells

Quiescent human fibroblast (hFB), human primary cancer associated fibroblast from three different CCA patients (hCAF 1, 2 and 3) and activated hepatic myofibroblasts (LX-2) were plated onto multiwell plates and grown to approximately 70% confluency. Cells were treated as indicated with increasing doses of navitoclax for 48 hrs. Cells were analyzed for apoptotic nuclear morphology by DAPI-staining and quantitation of apoptotic nuclei by fluorescence microscopy (panel A; mean ± SEM; n=3; ** p≤0.01). hFB, hCAF, LX2 as well as the human CCA cell lines HuCCT-1, Mz-ChA-1, KMCH and KMBC were treated with navitoclax (1μM) or vehicle for 48 hrs. Apoptosis was measured by DAPI-staining with quantitation of apoptotic nuclei by fluorescence microscopy (panel B, upper graph; mean ± SEM; n=3; ** p≤0.01) or fluorometric analysis of caspase 3/7 activity displayed as fold change compared to vehicle control (panel B, lower graph; mean ± SEM; n≥5; * p≤0.05).

Figure 2. Activated hepatic myofibroblasts exhibit alterations in their Bcl-2 protein profile and are sensitized to navitoclax by Bax

Whole cell lysates were prepared from quiescent hFB, hepatic myofibroblasts LX-2 and hCAF as well as quiescent rat fibroblasts (rFB), rat cancer associated fibroblasts (rCAF) and the malignant erbB-2/neu transformed rat cholangiocyte cell line (BDEneu) that is employed in the described in-vivo model of cholangiocarcinoma. Cell lysates were subject to immunoblot analysis of Bcl-2 proteins. Except where indicated by white lines, all lanes were adjacent on the membranes; in some cases, additional lanes originally run between those shown are omitted for clarity (panels A and B). All full-length blots/gels are presented in Supplementary Figure 6. hFB, LX-2 and hCAF cells were grown to approximately 50% confluency on glass chamber slides and treated with navitoclax (1μM) for the indicated time. Cells were then analyzed by fluorescence microscopy using a conformation-specific antibody (6A7) against activated Bax. Bax positive cells are plotted as percentage of all cells (panel C; mean ± SEM; n=4; ** p≤0.01). Wild-type LX-2 cells as well as stably transfected shBax and shBak LX-2 cells were treated with navitoclax (1μM, 24h) and apoptosis was assessed by DAPI staining and fluorescence microscopy (panel D upper graph; mean ± SEM; n=4; ** p≤0.01) as well as fluorometric measurement of caspase 3/7 activity (panel D lower graph; mean ± SEM; n=6; ** p≤0.01)

Figure 3. Knockdown of Mcl-1 in CCA cells induces navitoclax sensitivity while Mcl-1 overexpression in LX-2 cells confers resistance to navitoclax induced apoptosis

Mcl-1 was knocked down in the human CCA cell line KMCH by shRNA technique (Western blot for Mcl-1, see panel A inset) and cells were treated with navitoclax (1 μM) or vehicle for 48 hrs. Apoptosis was measured by DAPI-staining and fluorescence microscopy (panel A, upper graph; mean ± SEM; n=3; ** p≤0.01) or fluorometric analysis of caspase 3/7 activity displayed as fold change compared to vehicle control (panel A, lower graph; mean ± SEM; n≥5; ** p≤0.001). Mcl-1 was overexpressed in the activated hepatic myofibroblast LX-2 (Western blot for Mcl-1, see panel B inset) and cells were treated with navitoclax (1 μM or 5 μM) or vehicle for 48 hrs. Apoptosis was assessed by DAPI-staining and measurement of caspase 3/7 activity as described above ( mean ± SEM; n=3; ** p≤0.01)

Figure 4. Navitoclax selectively induces release of mitochondrial proapoptotic factors in activated myofibroblasts

Heavy membranes enriched in mitochondria were isolated from LX-2 cells and incubated with navitoclax (10 μM) or vehicle. Mitochondria were separated from supernatant by centrifugation and fractions were subject to immunoblot analysis to assay for Smac release from the mitochondria into the supernatant (panel A). LX-2 cells were treated with navitoclax (1 μM) and separated into cytoplasmic and mitochondria-containing fractions (pellet - loading control) by differential centrifugation after selective digitonin permeabilization. Fractions were analyzed for Smac and cytochrome C by immunoblot (panel B). Cytoplasmic and mitochondria-containing fractions (pellet) of shBax and shBak LX-2 cells and human CCA cell lines MzChA-1 and HuCCT-1 treated with navitoclax (1 μM) are shown in panels C and D, respectively. Note in these short-term incubation studies, the pool of cytochrome C and/or Smac released was limited and did not significantly influence the amount of these proteins in the pellet. hFB, LX-2, shBax LX-2 and Mz-ChA-1 CCA cells were treated with navitoclax or vehicle for 24 hrs. After tetramethylrhodamine methyl ester (TMRM, 100 nM) loading, cells were analyzed for mitochondrial depolarization and subsequent loss of fluorescence by flow cytometry. TMRM fluorescence (excitation wavelength 544 nM) intensity was measured in 50,000 cells (panel E).

Figure 5. Activated myofibroblasts display increased localization of proapoptotic BH3 only proteins to mitochondria and binding to anti-apoptotic Bcl-2 proteins

Mitochondria from LX-2, hCAF and human CCA cells were isolated by nitrogen cavitation and subject to immunoblot analysis for pro-apoptotic Bcl-2 family proteins (panel A). Immunoprecipitation of Bcl-X_L from cell lysates of LX-2 cells and subsequent immunoblot analysis for the pro-apoptotic BH3 only protein Bim is shown in panel B. non-specific rabbit IgG was used as negative control during the immunoprecipitation (panel B).

Figure 6. Navitoclax depletes CAF from BDEneu tumors and reduces characteristic tumor stroma ECM

Immunofluorescence double stainings for α-SMA (red) and cytokeratin 7 (CK-7, green; panel A, upper image, 40×) as well as tenascin C (Ten C, green) and cytokeratin 7 (CK-7 red; panel A, lower image, 40×) were performed on representative sections of BDEneu tumors harvested 17 days after tumor cell implantation. Frozen sections of BDEneu tumors from animals treated with two doses of vehicle or navitoclax (5 mg/kg) were stained for α-SMA or cytokeratin 7 and co-labeled with the TUNEL staining. The number of α-SMA/TUNEL and CK-7/TUNEL positive cells was assessed and plotted as percentage of all α-SMA or CK-7 positive cells, respectively (panel B; mean ± SEM; n=4; * p≤0.05). BDEneu tumors from animals treated with two doses of navitoclax (5 mg/kg) were stained for α-SMA, tenascin C and cytokeratin 7. Confocal microphotographs of representative tumor areas were analyzed by digital morphometry and α-SMA and tenascin C positive areas were expressed as percent of total tumor area (panel C; mean ± SEM; n=8; ** p≤0.001). α-SMA, Ten C and CK-7 mRNA expression was quantified by real-time PCR and normalized to 18S rRNA. Expression of α-SMA and tenascin c mRNA was plotted as ratio to CK-7 to control for the quantity of tumor-stroma in the sample (panel D; mean ± SEM; n=7; * p≤0.05)

Figure 7. Navitoclax treatment reduces BDEneu tumor size and metastasis, improves survival and alters tumor composition

BDEneu tumors from animals treated with navitoclax (5 mg/kg) or vehicle for 10 days were carefully excised, remaining liver tissue was removed, then tumors were measured and weighted (panel A; mean ± SEM; n=8; * p≤0.05) and tumor-liver weight ratios were calculated (panel B; mean ± SEM; n=8; * p≤0.05). After removal of the liver, the peritoneal cavity was carefully examined for metastases (panel C; mean ± SEM; n=8; * p≤0.05). To assess effects of navitoclax on survival, animals were treated with navitoclax as described for 21 days and survival was documented (panel D; mean ± SEM; n=18; p≤0.05). Frozen tumor sections were stained for α-SMA, tenascin C and cytokeratin 7. Digital morphometry was performed on confocal microphotographs and ratio of α-SMA and tenascin C positive stroma to tumor area was calculated (panel E; mean ± SEM; n=4; ** p≤0.01). Representative photomicrographs of hematoxylin-eosin stained tumor sections are shown in panel F. Characteristic tumor areas are outlined.