Adjuvant therapy after resection of colorectal liver metastases: the predictive value of the MSKCC clinical risk score in the era of modern chemotherapy

Background: Despite introduction of effective chemotherapy protocols, it has remained uncertain, if patients with colorectal cancer (CRC) liver metastases should receive adjuvant therapy. Clinical or molecular predictors may help to select patients at high risk for disease recurrence and death who obtain a survival advantage by adjuvant chemotherapy. Methods: A total of 297 patients with potentially curative resection of CRC liver metastases were analyzed. These patients had no neoadjuvant therapy, no extrahepatic disease and negative resection margins. The primary endpoint was overall survival. Patients’ risk status was evaluated using the Memorial Sloan-Kettering Cancer Center clinical risk score (MSKCC-CRS). Multivariable analyses were performed using Cox proportional hazard models. Results: A total of 137 (43%) patients had a MSKCC-CRS > 2. Adjuvant chemotherapy was administered to 116 (37%) patients. Patients who received adjuvant chemotherapy were of younger age (p = 0.03) with no significant difference in the presence of multiple metastases (p = 0.72) or bilobar metastases (p = 0.08). On multivariate analysis adjuvant chemotherapy was associated with improved survival in the entire cohort (Hazard ratio 0.69; 95% confidence interval 0.69–0.98). It improved survival markedly in high-risk patients with a MSKCC-CRS > 2 (HR 0.40; 95% CI 0.23–0.69), whereas it was of no benefit in patients with a MSKCC-CRS ≤ 2 (HR 0.90; 95% CI 0.57–1.43). Conclusions: The MSKCC-CRS offers a tool to select patients for adjuvant therapy after resection of CRC liver metastases. Validation in independent patient cohorts is required

Pan-Bcl-2 inhibitor Obatoclax is a potent late stage autophagy inhibitor in colorectal cancer cells independent of canonical autophagy signaling

Background: Colorectal cancer is the third most common malignancy in humans and novel therapeutic approaches are urgently needed. Autophagy is an evolutionarily highly conserved cellular process by which cells collect unnecessary organelles or misfolded proteins and subsequently degrade them in vesicular structures in order to refuel cells with energy. Dysregulation of the complex autophagy signaling network has been shown to contribute to the onset and progression of cancer in various models. The Bcl-2 family of proteins comprises central regulators of apoptosis signaling and has been linked to processes involved in autophagy. The antiapoptotic members of the Bcl-2 family of proteins have been identified as promising anticancer drug targets and small molecules inhibiting those proteins are in clinical trials. Methods: Flow cytometry and colorimetric assays were used to assess cell growth and cell death. Long term 3D cell culture was used to assess autophagy in a tissue mimicking environment in vitro. RNA interference was applied to modulate autophagy signaling. Immunoblotting and q-RT PCR were used to investigate autophagy signaling. Immunohistochemistry and fluorescence microscopy were used to detect autophagosome formation and autophagy flux. Results: This study demonstrates that autophagy inhibition by obatoclax induces cell death in colorectal cancer (CRC) cells in an autophagy prone environment. Here, we demonstrate that pan-Bcl-2 inhibition by obatoclax causes a striking, late stage inhibition of autophagy in CRC cells. In contrast, ABT-737, a Mcl-1 sparing Bcl-2 inhibitor, failed to interfere with autophagy signaling. Accumulation of p62 as well as Light Chain 3 (LC3) was observed in cells treated with obatoclax. Autophagy inhibition caused by obatoclax is further augmented in stressful conditions such as starvation. Furthermore, our data demonstrate that inhibition of autophagy caused by obatoclax is independent of the essential pro-autophagy proteins Beclin-1, Atg7 and Atg12. Conclusions: The objective of this study was to dissect the contribution of Bcl-2 proteins to autophagy in CRC cells and to explore the potential of Bcl-2 inhibitors for autophagy modulation. Collectively, our data argue for a Beclin-1 independent autophagy inhibition by obatoclax. Based on this study, we recommend the concept of autophagy inhibition as therapeutic strategy for CRC

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

BACKGROUND: Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro. METHODS: RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively. RESULTS: Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition. CONCLUSION: Our data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies

TRAIL-induced apoptosis of hepatocellular carcinoma cells isaugmented by targeted therapies

AIM: To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors, in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), on overcoming TRAIL resistance in hepatocellular carcinoma (HCC) and to study the efficacy of agonistic TRAIL antibodies, as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. METHODS: Surface expression of TRAIL receptors (TRAIL-R1-4) and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) were analyzed by flow cytometry and Western blotting, respectively. Knock-down of MCL-1 and BCL-x(L) was performed by transfecting specific small interfering RNAs. HCC cells were treated with kinase inhibitors and chemotherapeutic drugs. Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. RESULTS: TRAIL-R1 and -R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However, treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates. Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002 [inhibition of phosphoinositol-3-kinase (PI3K)], AG1478 (epidermal growth factor receptor kinase), PD98059 (MEK1), rapamycin (mammalian target of rapamycin) and the multi-kinase inhibitor Sorafenib. Furthermore, the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) play a major role in TRAIL resistance: knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells. Additionally, knock-down of MCL-1 and BCL-x(L) led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K. CONCLUSION: Our data identify the blockage of survival kinases, combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC

Beyond cell death - antiapoptotic Bcl-2 proteins regulate migration and invasion of colorectal cancer cells in vitro.

Migration and invasion of malignant cells are prerequisites for cancer progression and metastasis. The Bcl-2 family of proteins consists of about 25 members and has been extensively studied in the context of apoptosis. Despite the fact that small molecules targeting Bcl-2 proteins have already entered clinical trials, very few studies investigated a role of antiapoptotic Bcl-2 proteins beside cell death in the context of metastasis. The aim of this study was to dissect a potential role of the antiapoptotic Bcl-2 proteins Mcl-1, Bcl-2 and Bcl-xL on migration and invasion of colorectal cancer cells independent of their cell death control function. We used migration and invasion assays as well as three dimensional cell cultures to analyze colorectal cancer cell lines (HT29 and SW480) after siRNA mediated knockdown or overexpression of Mcl-1, Bcl-2 or Bcl-xL. We observed neither spontaneous cell death induction nor impaired proliferation of cells lacking Mcl-1, Bcl-2 or Bcl-xL. In contrast, knockdown of Mcl-1 led to increased proliferation. Strikingly, we demonstrate a profound impairment of both, migration and invasion, of colorectal cancer cells after Mcl-1, Bcl-2 or Bcl-xL knockdown. This phenotype was completely revised in cells overexpressing Mcl-1, Bcl-2 or Bcl-xL. The most pronounced effect among the investigated proteins was observed for Bcl-2. The data presented indicate a pivotal role of Mcl-1, Bcl-2 and Bcl-xL for migration and invasion of colorectal cancer cells independent of their known antiapoptotic effects. Thus, our study illustrates novel antitumoral mechanisms of Bcl-2 protein targeting

Bcl-xL is an oncogenic driver in colorectal cancer

Colorectal cancer (CRC) is the second most common malignant neoplasia in women and men worldwide. The B-cell lymphoma 2 (Bcl-2) protein family is mainly known for its pivotal role in the regulation of the mitochondrial death pathway. Anti-apoptotic Bcl-2 proteins may provide survival benefits and induce therapy resistance in cancer cells. Among anti-apoptotic Bcl-2 proteins, we found solely Bcl-xL strongly upregulated in human CRC specimens. In order to study protein function in the context of tumor initiation and progression in vivo, we generated a mouse model lacking Bcl-xL in intestinal epithelial cells (Bcl-xL(IEC-KO)). If challenged in an inflammation-driven tumor model, Bcl-xL(IEC-KO) mice showed a significantly reduced tumor burden with lower tumor numbers per animal and decreased tumor sizes. Analysis of cell death events by immunohistochemistry and immunoblotting revealed a striking increase of apoptosis in Bcl-xL-negative tumors. qRT-PCR and immunohistochemistry excluded changes in proliferative capacity and immune cell infiltration as reasons for the reduced tumor load and thereby identify apoptosis as key mechanism. Human CRC tissue was cultured ex vivo and treated with the small molecule compound ABT-737, which inhibits Bcl-xL and Bcl-2. Under ABT-737 treatment, the amount of apoptotic tumor cells significantly increased compared with controls, whereas proliferation levels remained unaltered. In summary, our findings identify Bcl-xL as a driver in colorectal tumorigenesis and cancer progression, making it a valuable target for clinical application