Systems biology approaches to develop innovative strategies for lung cancer therapy.

Lung cancer (LC) is a number one killer of cancer-related death among men and women worldwide. Major advances have been made in the diagnosis, staging and use of surgery for LC, but systemic chemotherapy and radiotherapy alone or in combination with some targeted agents remains the core treatment of advanced LC. Unfortunately, in spite of improved diagnosis, surgical methods and new treatments, mortality is still extremely high among LC patients. To understand the precise functioning of signaling pathways associated with resistance to current treatments in LC, as well as to identify novel treatment regimens, a holistic approach to analyze signaling networks should be applied. Here, we describe systems biology-based approaches to generate biomarkers and novel therapeutic targets in LC, as well as how this may contribute to personalized treatment for this malignancy.

Harnessing the lysosome-dependent antitumor activity of phenothiazines in human small cell lung cancer.

Phenothiazines are a family of heterocyclic compounds whose clinical utility includes treatment of psychiatric disorders as well as chemotherapy-induced emesis. Various studies have demonstrated that these compounds possess cytotoxic activities in tumor cell lines of different origin. However, there is considerable confusion regarding the molecular basis of phenothiazine-induced cell death. Lung cancer (LC) remains one of the most prevalent and deadly malignancies worldwide despite considerable efforts in the development of treatment strategies, especially new targeted therapies. In this work, we evaluated the potential utility of phenothiazines in human LC. We show that phenothiazines as single treatment decreased cell viability and induced cell death preferentially in small cell lung carcinoma (SCLC) over non small cell lung carcinoma (NSCLC) cell lines. Sensitivity to phenothiazines was not correlated with induction of apoptosis but due to phenothiazine-induced lysosomal dysfunction. Interestingly, the higher susceptibility of SCLC cells to phenothiazine-induced cell death correlated with an intrinsically lower buffer capacity in response to disruption of lysosomal homeostasis. Importantly, this effect in SCLC occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. Our data thus uncovered a novel context-dependent activity of phenothiazines in SCLC and suggest that phenothiazines could be considered as a treatment regimen of this disease, however, extended cell line analyses as well as in vivo studies are needed to make such conclusion.

Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest.

Increasing evidence suggests that tumor-initiating cells (TICs), also called cancer stem cells, are partly responsible for resistance to DNA-damaging treatment. Here we addressed if such a phenotype may contribute to radio- and cisplatin resistance in non-small cell lung cancer (NSCLC). We showed that four out of eight NSCLC cell lines (H125, A549, H1299 and H23) possess sphere-forming capacity when cultured in stem cell media and three of these display elevated levels of CD133. Indeed, sphere-forming NSCLC cells, hereafter called TICs, showed a reduced apoptotic response and increased survival after irradiation (IR), as compared with the corresponding bulk cell population. Decreased cytotoxicity and apoptotic signaling manifested by diminished poly (ADP-ribose) polymerase (PARP) cleavage and caspase 3 activity was also evident in TICs after cisplatin treatment. Neither radiation nor cisplatin resistance was due to quiescence as H125 TICs proliferated at a rate comparable to bulk cells. However, TICs displayed less pronounced G2 cell cycle arrest and S/G2-phase block after IR and cisplatin, respectively. Additionally, we confirmed a cisplatin-refractory phenotype of H125 TICs in vivo in a mouse xenograft model. We further examined TICs for altered expression or activation of DNA damage repair proteins as a way to explain their increased radio- and/or chemotherapy resistance. Indeed, we found that TICs exhibited increased basal γH2AX (H2A histone family, member X) expression and diminished DNA damage-induced phosphorylation of DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia-mutated (ATM), Krüppel-associated protein 1 (KAP1) and monoubiquitination of Fanconi anemia, complementation group D2 (FANCD2). As a proof of principle, ATM inhibition in bulk cells increased their cisplatin resistance, as demonstrated by reduced PARP cleavage. In conclusion, we show that reduced apoptotic response, altered DNA repair signaling and cell cycle perturbations in NSCLC TICs are possible factors contributing to their therapy resistance, which may be exploited for DNA damage-sensitizing purposes.

Inhibition of Ephrin B3-mediated survival signaling contributes to increased cell death response of non-small cell lung carcinoma cells after combined treatment with ionizing radiation and PKC 412.

Radiation therapy is frequently used to treat non-small cell lung cancers (NSCLCs). We have previously shown that a combination of ionizing radiation (IR) and the staurosporine analog PKC 412, but not Ro 31-8220, increases cell death in NSCLC cells. To identify genes involved in the enhancement of cell death, a total gene profiling in response to co-administration of (i) PKC 412 with IR, or (ii) Ro 31-8220 with IR was implemented. These combined treatments caused upregulation of 140 and 179 genes and downregulation of 253 and 425 genes, respectively. Certain genes were selected and verified by real-time quantitative PCR and, of these genes, robust suppression of Ephrin B3 expression was suggested as a possible cell death-inducing mechanism of combined treatment with IR and PKC 412. Indeed, silencing of Ephrin B3 using siRNA in NSCLC cells resulted in a major alteration of their morphology with an elongated phenotype, decreased proliferation and increased cell death signaling. Moreover, silencing of Ephrin B3 in combination with IR caused a decrease in IR-mediated G(2)-arrest, induced cellular senescence, inhibited MAPK ERK and p38 phosphorylation, and caused an upregulation of p27(kip1) expression. Finally, silencing of Ephrin B3 in combination with IR sensitized U-1810 cells to IR-induced apoptosis. In conclusion, we identify and describe Ephrin B3 as a putative signaling molecule involved in the response of NSCLC cells to combined treatment with PKC 412 and ionizing radiation.

miRNA-214 modulates radiotherapy response of non-small cell lung cancer cells through regulation of p38MAPK, apoptosis and senescence.

BACKGROUND: Radio- and chemotherapy (RT/CT) resistance hampers success in combating small and non-small cell lung cancers (SCLC/NSCLC). The underlying molecular mechanisms of RT/CT resistance of LCs are multifactorial and have been understood in part hitherto. miRNAs, key regulators of mRNAs, are well-recognised oncomirs; however, their role in regulating RT response remains poorly understood. METHODS: Six human NSCLC and five SCLC cell lines with different SF2 values were investigated. Using microarray we examined whether expression of miRNAs is linked to the RT resistance of NSCLCs or SCLCs. Obtained data were validated by quantitative real-time PCR. Apoptosis and senescence were analysed using siRNA transfection, western blot and flow cytometry. RESULTS: miRNA-21, miRNA-1827, miRNA-214, miRNA-339-5p, miRNA-625, miRNA-768-3p, miRNA-523-3p, miRNA-1227, miRNA-324-5p, miRNA-423-3p, miRNA-1301 and miRNA-1249 are differentially expressed in LC cells. miRNA-214 is upregulated in RT-resistant NSCLC cells relative to radiosensitive counterparts. Considering miRNA-214 as a putative regulator of RT resistance, we demonstrate that knockdown of miRNA-214 in radioresistant NSCLCs sensitised them to RT by stimulation of senescence. Consistently, overexpression of miRNA-214 in radiosensitive NSCLCs protected against RT-induced apoptosis. Protection was mediated by p38MAPK, as downregulation of this kinase could reverse the miRNA-214 overexpression-induced resistance of NSCLC cells. CONCLUSION: miRNA profiling of LC revealed putative RT resistance signalling circuits, which might help in sensitisation of LC to RT.

Chemosensitization by phenothiazines in human lung cancer cells: impaired resolution of γH2AX and increased oxidative stress elicit apoptosis associated with lysosomal expansion and intense vacuolation.

Chemotherapy resistance poses severe limitations on the efficacy of anti-cancer medications. Recently, the notion of using novel combinations of 'old' drugs for new indications has garnered significant interest. The potential of using phenothiazines as chemosensitizers has been suggested earlier but so far our understanding of their molecular targets remains scant. The current study was designed to better define phenothiazine-sensitive cellular processes in relation to chemosensitivity. We found that phenothiazines shared the ability to delay γH2AX resolution in DNA-damaged human lung cancer cells. Accordingly, cells co-treated with chemotherapy and phenothiazines underwent protracted cell-cycle arrest followed by checkpoint escape that led to abnormal mitoses, secondary arrest and/or a form of apoptosis associated with increased endogenous oxidative stress and intense vacuolation. We provide evidence implicating lysosomal dysfunction as a key component of cell death in phenothiazine co-treated cells, which also exhibited more typical hallmarks of apoptosis including the activation of both caspase-dependent and -independent pathways. Finally, we demonstrated that vacuolation in phenothiazine co-treated cells could be reduced by ROS scavengers or the vacuolar ATPase inhibitor bafilomycin, leading to increased cell viability. Our data highlight the potential benefit of using phenothiazines as chemosensitizers in tumors that acquire molecular alterations rendering them insensitive to caspase-mediated apoptosis.

Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86.

BACKGROUND: Radiotherapy is central in the treatment of cervical cancer. The formation of DNA double-strand breaks is considered to be critical for the radiotherapeutic effect. The non-homologous end joining (NHEJ) proteins DNA-PKcs, Ku70 and Ku86 have a major role in repairing DNA lesions. The objective of this study was to analyse if the expression of DNA-PKcs, Ku70 and Ku86 and their downstream signalling molecules p53, p21 and Mdm-2 are altered in residual cervical tumours after radiotherapy. METHODS: Retrospective analysis of 127 patients with cervical cancer stage IB-IIA treated with preoperative radiotherapy and radical surgery, revealed residual tumour in the cervical specimen in 30 patients. In 22 cases tumour material from residual and corresponding primary tumour were retrieved and the expression of DNA-PKcs, Ku86, Ku70, p53, p21 and Mdm-2 were assessed by immunohistochemistry. RESULTS: Residual tumours showed increased frequency of DNA-PKcs (P=0.037), Ku70 (P=0.018), Ku86 (P=0.008) positive cells. A correlation in DNA-PKcs expression between primary and residual tumours was found. The frequency of p21-positive cells was decreased (P=0.007) in residual tumours whereas no change in p53 or Mdm-2-positive cells were observed. CONCLUSION: Our results show that cervical carcinoma surviving radiotherapy have an increased DNA-PK expression. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of DNA-PK function may be part of a radioresistance mechanism within this tumour type.

Cisplatin induces the proapoptotic conformation of Bak in a deltaMEKK1-dependent manner.

In a panel of four human melanoma cell lines, equitoxic doses of cisplatin induced the proapoptotic conformation of the Bcl-2 family protein Bak prior to the execution phase of apoptosis. Because cisplatin-induced modulation of the related Bax protein was seen in only one cell line, a degree of specificity in the signal to Bak is indicated. Little is known about upstream regulation of Bak activity. In this study, we examined whether the apoptosis-specific pathway mediated by a kinase fragment of MEKK1 (DeltaMEKK1) is involved in the observed Bak modulation. We report that expression of a kinase-inactive fragment of MEKK1 (dominant negative MEKK [dnMEKK]) efficiently blocked cisplatin-induced modulation of Bak and cytochrome c release and consequently also reduced DEVDase activation and nuclear fragmentation. Accordingly, expression of a kinase-active MEKK1 fragment (dominant positive MEKK) was sufficient to induce modulation of Bak in three cell lines and to induce apoptosis in two of these. dnMEKK did not block cisplatin-induced c-Jun N-terminal kinase (JNK) activation, in agreement with a specifically proapoptotic role for the DeltaMEKK1 pathway. Finally, we show that reduction of Bak expression by antisense Bak reduced cisplatin-induced loss of mitochondrial integrity and caspase cleavage activity in breast cancer cell lines. In summary, we have identified Bak as a cisplatin-regulated component downstream in a proapoptotic, JNK-independent DeltaMEKK1 pathway.

The MEK1 inhibitor PD98059 sensitizes C8161 melanoma cells to cisplatin-induced apoptosis.

The regulation of apoptosis is believed to be dependent on the balance of the activities of different intracellular signalling systems. Activation of the SAPK/JNK pathway is implied in pro-apoptotic signalling, while activation of the MEK1/ERK pathway may have a viability-promoting effect. We show here that treatment with the MEK1 inhibitor PD98059 sensitizes the human melanoma cell line C8161 to cisplatin-induced apoptosis. In these cells, cisplatin at 40 microM did not elicit significant cell death, whereas massive cell death was seen when cells were pretreated for 20 h with 40 microM PD98059 before the addition of cisplatin. Concomitant addition of PD98059 and cisplatin did not have any sensitizing effect, and PD98059 on its own did not induce apoptosis. However, in three other human melanoma cell lines PD98059 did not potentiate cisplatin-induced apoptosis. Instead, in one of these cell lines (AA), PD98059 protected against cisplatin-induced cytotoxicity. We conclude that blocking of the MEK1/ERK pathway may, in some instances, potentiate the cytotoxic effect of cisplatin on human melanoma cell lines, whereas in other instances it may have a protective effect. Thus it cannot be regarded as a general approach to sensitizing melanoma cells to drug-induced apoptosis.

Increased apoptosis and increased clonogenic survival of 12V-H-ras transformed rat fibroblasts in response to cisplatin.

Mutationally activated Ras is involved in tumor progression and likely also in drug resistance. Using survival, viability and apoptosis assays, we have here compared the cisplatin sensitivities of FR3T3 rat fibroblasts and a 12V-H-ras transformed subline (Ras2:3). Around 24 h after cisplatin treatment Ras2:3 cells showed higher apoptosis levels and lower viability than FR3T3. This increased sensitivity correlated with weaker cisplatin-induced activation of Jun N-terminal kinase (JNK). In contrast to apoptosis assays, colony formation assays showed that Ras2:3 were more resistant to cisplatin than were FR3T3. This was partly due to the increased cisplatin sensitivity of FR3T3 seeded at low densities, as required in colony formation assays. In addition, Ras2:3 cisplatin survivors had a higher relative proliferative capacity. Cell cycle analyses showed that FR3T3 cells initially responded with a dose-dependent G2 arrest, while Ras2:3 accumulated in S-phase. Experiments with an anti-apoptotic mutant of MEKK1 suggested that the apoptotic response of Ras2:3 cells is not specific to the S-phase fraction. In summary, the cisplatin response of ras-transformed fibroblasts is distinct from that of parental cells, in that they show increased apoptosis, a different cell cycle response and increased post-treatment proliferative capacity. The results illustrate the need to carefully consider methods and protocols for in vitro studies on chemotherapy sensitivity.