ABSTRACT
Tumor hypoxia plays an important role in controlling tumor progression through signaling pathways related to the transcription factor HIF-1. In addition to enhancing migration, promoting angiogenesis and regulating metabolism, the hypoxic environment also affects immune function. In this hypoxic microenvironment an immunosuppressive milieu is established, where HIF-1 upregulates the expression of PD-L1, a key regulator of the immune response. We have found that elevated expression of PD-L1 correlates with increased HIF-1 levels in cancer cell lines and clinical samples. Thus, the co-inhibition of HIF-1 and PD-1/PD-L1 offers promising therapeutic possibilities. In this review we have examined the limitations of HIF-1 and PD-1/PD-L1 inhibition as monotherapy, explored their combined benefits and evaluated the feasibility of targeting PD-L1 with HIF-1 inhibitors.
Subject(s)
B7-H1 Antigen , Neoplasms , Programmed Cell Death 1 Receptor , Tumor Hypoxia , Tumor Microenvironment , Humans , Tumor Microenvironment/immunology , Tumor Microenvironment/drug effects , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/metabolism , Neoplasms/immunology , Neoplasms/drug therapy , Neoplasms/pathology , Tumor Hypoxia/drug effects , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Signal Transduction/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/metabolismABSTRACT
BACKGROUND: Inhibition of mutant KRAS challenged cancer research for decades. Recently, allele-specific inhibitors were approved for the treatment of KRAS-G12C mutant lung cancer. However, de novo and acquired resistance limit their efficacy and several combinations are in clinical development. Our study shows the potential of combining G12C inhibitors with farnesyl-transferase inhibitors. METHODS: Combinations of clinically approved farnesyl-transferase inhibitors and KRAS G12C inhibitors are tested on human lung, colorectal and pancreatic adenocarcinoma cells in vitro in 2D, 3D and subcutaneous xenograft models of lung adenocarcinoma. Treatment effects on migration, proliferation, apoptosis, farnesylation and RAS signaling were measured by histopathological analyses, videomicroscopy, cell cycle analyses, immunoblot, immunofluorescence and RAS pulldown. RESULTS: Combination of tipifarnib with sotorasib shows synergistic inhibitory effects on lung adenocarcinoma cells in vitro in 2D and 3D. Mechanistically, we present antiproliferative effect of the combination and interference with compensatory HRAS activation and RHEB and lamin farnesylation. Enhanced efficacy of sotorasib in combination with tipifarnib is recapitulated in the subcutaneous xenograft model of lung adenocarcinoma. Finally, combination of additional KRAS G1C and farnesyl-transferase inhibitors also shows synergism in lung, colorectal and pancreatic adenocarcinoma cellular models. DISCUSSION: Our findings warrant the clinical exploration of KRAS-G12C inhibitors in combination with farnesyl-transferase inhibitors.
Subject(s)
Adenocarcinoma of Lung , Adenocarcinoma , Colorectal Neoplasms , Lung Neoplasms , Pancreatic Neoplasms , Humans , Animals , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Transferases , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , MutationABSTRACT
Malignant melanoma is challenging to treat, and metastatic cases need chemotherapy strategies. Targeted inhibition of commonly mutant BRAF V600E by inhibitors is efficient but eventually leads to resistance and progression in the vast majority of cases. Numerous studies investigated the mechanisms of resistance in melanoma cell lines, and an increasing number of in vivo or clinical data are accumulating. In most cases, bypassing BRAF and resulting reactivation of the MAPK signaling, as well as alternative PI3K-AKT signaling activation are reported. However, several unique changes were also shown. We developed and used a patient-derived tumor xenograft (PDTX) model to screen resistance evolution in mice in vivo, maintaining tumor heterogeneity. Our results showed no substantial activation of the canonical pathways; however, RNAseq and qPCR data revealed several altered genes, such as GPR39, CD27, SLC15A3, IFI27, PDGFA, and ABCB1. Surprisingly, p53 activity, leading to apoptotic cell death, was unchanged. The found biomarkers can confer resistance in a subset of melanoma patients via immune modulation, microenvironment changes, or drug elimination. Our resistance model can be further used in testing specific inhibitors that could be used in future drug development, and combination therapy testing that can overcome inhibitor resistance in melanoma.
Subject(s)
Melanoma , Proto-Oncogene Proteins B-raf , Vemurafenib , Animals , Humans , Mice , Cell Line, Tumor , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Phosphatidylinositol 3-Kinases/genetics , Receptors, G-Protein-Coupled/genetics , Vemurafenib/pharmacology , Vemurafenib/therapeutic useABSTRACT
Chemotherapy resistance in tumours is due to complex processes and is responsible for about half of all cancer deaths. In my thesis, I have investigated multiple different resistance mechanisms, most in depth the effect of multidrug resistance (MDR) caused by expression and function of P-glycoprotein (Pgp), and the MDR-selective compounds (such as NSC297366) effectively targeting it. The mechanism was investigated using cell models with different Pgp expression. Seeking the mechanism of action of the MDR-selective NSC297366, we showed that the intracellular iron-binding chelator molecule is able to reduce the amount of free iron available within the cell. Furthermore, by active efflux through Pgp in MDR cells, the compounds can lead to intracellular iron deficiency, upregulation of iron-demanding processes such as cell cycle and apoptosis, and selective death of MDR cancer cells. Our results raise the possibility of targeted killing of MDR phenotypic cancer cells resistant to other therapies, which in combination with conventional chemotherapeutic approaches may form the basis of a strategy of long-term control of the disease.
Subject(s)
Drug Resistance, Neoplasm , Neoplasms , Humans , Neoplasms/drug therapy , Drug Resistance, Multiple , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/pharmacology , Iron/metabolism , Iron/pharmacology , Iron/therapeutic useABSTRACT
Background. The dual role of GCs has been observed in breast cancer; however, due to many concomitant factors, GR action in cancer biology is still ambiguous. In this study, we aimed to unravel the context-dependent action of GR in breast cancer. Methods. GR expression was characterized in multiple cohorts: (1) 24,256 breast cancer specimens on the RNA level, 220 samples on the protein level and correlated with clinicopathological data; (2) oestrogen receptor (ER)-positive and -negative cell lines were used to test for the presence of ER and ligand, and the effect of the GRß isoform following GRα and GRß overexpression on GR action, by in vitro functional assays. Results. We found that GR expression was higher in ER- breast cancer cells compared to ER+ ones, and GR-transactivated genes were implicated mainly in cell migration. Immunohistochemistry showed mostly cytoplasmic but heterogenous staining irrespective of ER status. GRα increased cell proliferation, viability, and the migration of ER- cells. GRß had a similar effect on breast cancer cell viability, proliferation, and migration. However, the GRß isoform had the opposite effect depending on the presence of ER: an increased dead cell ratio was found in ER+ breast cancer cells compared to ER- ones. Interestingly, GRα and GRß action did not depend on the presence of the ligand, suggesting the role of the "intrinsic", ligand-independent action of GR in breast cancer. Conclusions. Staining differences using different GR antibodies may be the reason behind controversial findings in the literature regarding the expression of GR protein and clinicopathological data. Therefore, caution in the interpretation of immunohistochemistry should be applied. By dissecting the effects of GRα and GRß, we found that the presence of the GR in the context of ER had a different effect on cancer cell behaviour, but independently of ligand availability. Additionally, GR-transactivated genes are mostly involved in cell migration, which raises GR's importance in disease progression.
Subject(s)
Breast Neoplasms , Glucocorticoids , Humans , Female , Glucocorticoids/pharmacology , Ligands , Protein IsoformsABSTRACT
The progressive accumulation of misfolded α-synuclein (α-syn) in the brain is widely considered to be causal for the debilitating clinical manifestations of synucleinopathies including, most notably, Parkinson's disease (PD). Immunotherapies, both active and passive, against α-syn have been developed and are promising novel treatment strategies for such disorders. To increase the potency and specificity of PD vaccination, we created the 'Win the Skin Immune System Trick' (WISIT) vaccine platform designed to target skin-resident dendritic cells, inducing superior B and T cell responses. Of the six tested WISIT candidates, all elicited higher immune responses compared to conventional, aluminum adjuvanted peptide-carrier conjugate PD vaccines, in BALB/c mice. WISIT-induced antibodies displayed higher selectivity for α-syn aggregates than those induced by conventional vaccines. Additionally, antibodies induced by two selected candidates were shown to inhibit α-syn aggregation in a dose-dependent manner in vitro. To determine if α-syn fibril formation could also be inhibited in vivo, WISIT candidate type 1 (CW-type 1) was tested in an established synucleinopathy seeding model and demonstrated reduced propagation of synucleinopathy in vivo. Our studies provide proof-of-concept for the efficacy of the WISIT vaccine technology platform and support further preclinical and clinical development of this vaccine candidate.
ABSTRACT
BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are among the most abundant malignancies worldwide. Patients with recurrent/metastatic disease undergo combination chemotherapy containing cetuximab, the monoclonal antibody used against the epidermal growth factor receptor (EGFR). Cetuximab augments the effect of chemotherapy; however, a significant number of patients show therapy resistance. The mechanism of resistance is yet to be unveiled, although extracellular alterations of the receptor have been reported, and their role in cetuximab failure has been proposed. AIMS: Here, we investigate possible effects of the multi-exon deletion variant (EGFRvIII), and the single nucleotide polymorphism EGFR R521K on cetuximab efficacy. RESULTS: Our results show that in HNSCC patients, the EGFRvIII allele frequency is under 1%; therefore, it cannot lead to common resistance. EGFR R521K, present in 42% of the patients, is investigated in vitro in four HNSCC cell lines (two wild-type and two heterozygous for EGFR R521K). While no direct effect is found to be related to the EGFR status, cells harboring R521K show a reduced sensitivity in ADCC experiments and in vivo xenograft experiments. However, this preclinical difference is not reflected in the progression-free or overall survival of HNSCC patients. Furthermore, NK cell and macrophage presence in tumors is not related to EGFR R521K. DISCUSSION: Our results suggest that EGFR R521K, unlike reported previously, is unable to cause cetuximab resistance in HNSCC patients; therefore, its screening before therapy selection is not justifiable.
ABSTRACT
Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Head and Neck Neoplasms/drug therapy , Mutation , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Squamous Cell Carcinoma of Head and Neck/drug therapy , Animals , Apoptosis , Cell Proliferation , Cetuximab/administration & dosage , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , ErbB Receptors/metabolism , Erlotinib Hydrochloride/administration & dosage , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/pathology , Humans , Indoles/administration & dosage , Mice , Mice, SCID , Piperazines/administration & dosage , Protein Kinase Inhibitors/pharmacology , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Sulfonamides/administration & dosage , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Zoledronic Acid/administration & dosageABSTRACT
Head and neck squamous cell carcinomas (HNSCC) take many lifes worldwide. Patients with recurrent/metastatic disease receive combination chemotherapy containing anti-EGFR antibody cetuximab. However, resistance often hurdles therapy. The mechanism is yet to unveil, although EGFR extracellular alterations and activity of c-Met signaling were accused. We investigated the effects of EGFR-vIII and EGFR-R521K on cetuximab efficacy in HNSCC in cellular, xenograft, and clinical setup. Furthermore, we investigated the efficacy of c-Met inhibition in HNSCC in vitro and in vivo. We showed that EGFR-vIII is very rare in HNSCC, while the common R521K polymorphism abolishes antibody-dependent cellular cytotoxicity and in vivo antitumor effect of cetuximab. This selectivity was not reflected in immunophenotype or survival data of HNSCC patients, suggesting a more complex mechanism behind. Interestingly, c-Met inhibitor SU11274 was more effective in cetuximab-resistant, EGFR R521K heterozygous cells and xenografts, raising the possible importance of simultaneous targeting of the two receptors.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Cetuximab/pharmacology , Drug Resistance, Neoplasm , ErbB Receptors/genetics , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Humans , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/genetics , Xenograft Model Antitumor AssaysABSTRACT
Metastatic melanoma is the most aggressive type of skin cancer. Previously, we identified the plasma membrane Ca2+ pump isoform 4b (PMCA4b or ATP2B4) as a putative metastasis suppressor in BRAF mutant melanoma cells. Metastasis suppressors are often downregulated in cancer, therefore, it is important to identify the pathways involved in their degradation. Here, we studied the role of p38 MAPK in PMCA4b degradation and its effect on melanoma metastasis. We found that activation of p38 MAPK induces internalization and subsequent degradation of PMCA4b through the endo/lysosomal system that contributes to the low PMCA4b steady-state protein level of BRAF mutant melanoma cells. Moreover, BRAF wild type cell models including a doxycycline-inducible HEK cell system revealed that p38 MAPK is a universal modulator of PMCA4b endocytosis. Inhibition of the p38 MAPK pathway markedly reduced migration, colony formation and metastatic activity of BRAF mutant cells in vitro partially through an increase in PMCA4b and a decrease in ß4 integrin abundance. In conclusion, our data suggest that the p38 MAPK pathway plays a key role in PMCA4b degradation and inhibition of this pathway-by increasing the stability of PMCA4b-may provide a potential therapeutic target for inhibition of melanoma progression and metastasis.
Subject(s)
Cell Movement/genetics , Melanoma/genetics , Melanoma/pathology , Mutation/genetics , Plasma Membrane Calcium-Transporting ATPases/metabolism , Proteolysis , Proto-Oncogene Proteins B-raf/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , Calcium/metabolism , Cell Cycle Checkpoints/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Proliferation/drug effects , Endocytosis/drug effects , Endosomes/drug effects , Endosomes/metabolism , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Lysosomes/drug effects , Lysosomes/metabolism , Melanoma/enzymology , Melanoma/ultrastructure , NF-kappa B/metabolism , Neoplasm Metastasis , Protein Kinase Inhibitors/pharmacology , Protein Stability/drug effects , Protein Transport/drug effects , Skin Neoplasms/enzymology , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Skin Neoplasms/ultrastructure , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathology , Tumor Stem Cell Assay , Up-Regulation/drug effects , Up-Regulation/genetics , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5'-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes.
Subject(s)
Deoxyribonucleotides/analysis , Software , Taq Polymerase , Exodeoxyribonucleases , Fluorescence , KineticsABSTRACT
Clinical evidence shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and, eventually, most tumors become resistant to all available therapies. We compiled a focused library consisting of >500 commercially available or newly synthetized 8-hydroxyquinoline (8OHQ) derivatives whose toxicity is paradoxically increased rather than decreased by the activity of P-glycoprotein (Pgp), a transporter conferring multidrug resistance (MDR). Here, we deciphered the mechanism of action of NSC297366 that shows exceptionally strong Pgp-potentiated toxicity. Treatment of cells with NSC297366 resulted in changes associated with the activity of potent anticancer iron chelators. Strikingly, iron depletion was more pronounced in MDR cells due to the Pgp-mediated efflux of NSC297366-iron complexes. Our results indicate that iron homeostasis can be targeted by MDR-selective compounds for the selective elimination of multidrug resistant cancer cells, setting the stage for a therapeutic approach to fight transporter-mediated drug resistance. SIGNIFICANCE: Modulation of the MDR phenotype has the potential to increase the efficacy of anticancer therapies. These findings show that the MDR transporter is a "double-edged sword" that can be turned against resistant cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Iron Chelating Agents/pharmacology , Iron/metabolism , Neoplasms/drug therapy , Oxyquinoline/pharmacology , ATP Binding Cassette Transporter, Subfamily B/metabolism , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Humans , Inhibitory Concentration 50 , Iron Chelating Agents/therapeutic use , Neoplasms/pathology , Oxyquinoline/analogs & derivatives , Oxyquinoline/therapeutic useABSTRACT
Aberrant activation of FMS-like tyrosine receptor kinase 3 (FLT3) is implicated in the pathogenesis of acute myeloid leukemia (AML) in 20-30% of patients. In this study we identified a highly selective (phenylethenyl)quinazoline compound family as novel potent inhibitors of the FLT3-ITD and FLT3-D835Y kinases. Their prominent effects were confirmed by biochemical and cellular proliferation assays followed by mice xenograft studies. Our modelling experiments and the chemical structures of the compounds predict the possibility of covalent inhibition. The most effective compounds triggered apoptosis in FLT3-ITD AML cells but had either weak or no effect in FLT3-independent leukemic and non-leukemic cell lines. Our results strongly suggest that our compounds may become therapeutics in relapsing and refractory AML disease harboring various ITD and tyrosine kinase domain mutations, by their ability to overcome drug resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Leukemia, Myeloid, Acute/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Molecular Structure , Mutation , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Structure-Activity Relationship , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Oncogenic activation of the epidermal growth factor receptor (EGFR) signaling pathway occurs in a variety of tumor types, albeit in human melanoma, the contribution of EGFR is still unclear. The potential role of EGFR was analyzed in four BRAF-mutant, one NRAS-mutant and one wild-type NRAS-BRAF-carrying human melanoma cell lines. We have tested clinically available reversible tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib, irreversible EGFR-TKI pelitinib and a reversible experimental compound PD153035 on in-vitro proliferation, apoptosis, migration as well as in-vivo metastatic colonization in a spleen-liver model. The presence of the intracellular domain of EGFR protein and its constitutive activity were demonstrated in all cell lines. Efficacies of EGFR-TKIs showed significant differences, and irreversible inhibition had the strongest antitumor potential. Compared with BRAF-mutant cells, wild-type BRAF was associated with relative resistance against gefitinib. In combination with gefitinib, selective mutant BRAF-inhibitor vemurafenib showed additive effect in all BRAF-mutant cell lines. Treatment of BRAF-mutant cells with gefitinib or pelitinib attenuated in-vitro cell migration and in-vivo colonization. Our preclinical data suggest that EGFR is a potential target in the therapy of BRAF-mutant malignant melanoma; however, more benefits could be expected from irreversible EGFR-TKIs and combined treatment settings.
Subject(s)
Antineoplastic Agents/therapeutic use , ErbB Receptors/antagonists & inhibitors , Melanoma/drug therapy , Vemurafenib/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Humans , Melanoma/genetics , Melanoma/pathology , Mice , Mice, SCID , Vemurafenib/pharmacologyABSTRACT
Success of cancer treatment is often hampered by the emergence of multidrug resistance (MDR) mediated by P-glycoprotein (ABCB1/Pgp). Doxorubicin (DOX) is recognized by Pgp and therefore it can induce therapy resistance in breast cancer patients. In this study our aim was to evaluate the susceptibility of the pegylated liposomal formulation of doxorubicin (PLD/Doxil®/Caelyx®) to MDR. We show that cells selected to be resistant to DOX are cross-resistant to PLD and PLD is also ineffective in an allograft model of doxorubicin-resistant mouse B-cell leukemia. In contrast, PLD was far more efficient than DOX as reflected by a significant increase of both relapse-free and overall survival of Brca1-/-;p53-/- mammary tumor bearing mice. Increased survival could be explained by the delayed onset of drug resistance. Consistent with the higher Pgp levels needed to confer resistance, PLD administration was able to overcome doxorubicin insensitivity of the mouse mammary tumors. Our results indicate that the favorable pharmacokinetics achieved with PLD can effectively overcome Pgp-mediated resistance, suggesting that PLD therapy could be a promising strategy for the treatment of therapy-resistant breast cancer patients.
Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Doxorubicin/analogs & derivatives , Leukemia, B-Cell/drug therapy , Mammary Neoplasms, Experimental/drug therapy , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/pharmacology , Disease-Free Survival , Doxorubicin/administration & dosage , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Female , Humans , Leukemia, B-Cell/pathology , Male , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/pharmacokinetics , Polyethylene Glycols/pharmacology , Survival RateABSTRACT
Tumor hypoxia promotes neoangiogenesis and contributes to the radio- and chemotherapy resistant and aggressive phenotype of cancer cells. However, the migratory response of tumor cells and the role of small GTPases regulating the organization of cytoskeleton under hypoxic conditions have yet to be established. Accordingly, we measured the proliferation, migration, RhoA activation, the mRNA and protein levels of hypoxia inducible factor-1alpha (HIF-1α) and three small G-proteins, Rac1, cdc42 and RhoA in a panel of five human tumor cell lines under normoxic and hypoxic conditions. Importantly, HT168-M1 human melanoma cells with high baseline migration capacity showed increased HIF-1α and small GTPases expression, RhoA activation and migration under hypoxia. These activities were blocked by anti- HIF-1α shRNA. Moreover, the in vivo metastatic potential was promoted by hypoxia mimicking CoCl2 treatment and reduced upon inhibition of HIF-1α in a spleen to liver colonization experiment. In contrast, HT29 human colon cancer cells with low migration capacity showed limited response to in vitro hypoxia. The expression of the small G-proteins decreased both at mRNA and protein levels and the RhoA activation was reduced. Nevertheless, the number of lung or liver metastatic colonies disseminating from orthotopic HT29 grafts did not change upon CoCl2 or chetomin treatment. Our data demonstrates that the hypoxic environment induces cell-type dependent changes in the levels and activation of small GTPases and results in varying migratory and metastasis promoting responses in different human tumor cell lines.
Subject(s)
Gene Expression , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Animals , Cell Hypoxia/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Cytoskeleton/metabolism , Disease Models, Animal , Gene Silencing , Heterografts , Humans , Hypoxia/genetics , Hypoxia/metabolism , Male , Mice , Neoplasm Metastasis , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Organ Specificity/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
BACKGROUND: In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor. Nitrogen-containing bisphosphonates inhibit prenylation of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines. RESULTS: We confirmed that zoledronic acid was unable to inhibit the prenylation of mutant K-Ras unlike in the case of wild type K-Ras. In case of in vitro proliferation, the KRAS-mutant human NSCLC cell lines showed resistance to zoledronic acid wild-type KRAS-cells proved to be sensitive. Combinatory application of zoledronic acid enhanced the cytostatic effect of cisplatin. Zoledronic acid did not induce significant apoptosis. In xenograft model, zoledronic acid significantly reduced the weight of wild type KRAS-EGFR-expressing xenograft tumor by decreasing the proliferative capacity. Futhermore, zoledronic acid induced VEGF expression and improved in vivo tumor vascularization. MATERIALS AND METHODS: Membrane association of K-Ras was examined by Western-blot. In vitro cell viability, apoptotic cell death and migration were measured in NSCLC lines with different molecular background. The in vivo effect of zoledronic acid was investigated in a SCID mouse subcutaneous xenograft model. CONCLUSIONS: The in vitro and in vivo inhibitory effect of zoledronic acid was based on the blockade of cell cycle in wild type KRAS-expressing human NSCLC cells. The zoledronic acid induced vascularization supported in vivo cytostatic effect. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Diphosphonates/pharmacology , Imidazoles/pharmacology , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , A549 Cells , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cisplatin/pharmacology , Dose-Response Relationship, Drug , Genetic Predisposition to Disease , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice, Inbred BALB C , Mice, SCID , Neoplasm Invasiveness , Neovascularization, Pathologic , Phenotype , Signal Transduction/drug effects , Tumor Burden/drug effects , Vascular Endothelial Growth Factor A/metabolism , Xenograft Model Antitumor Assays , Zoledronic AcidABSTRACT
Lung cancer places a significant socio-economic burden on the Hungarian population. This overview summarizes the findings of collaborative translational lung cancer research efforts of three Hungarian flagship academic institutions, the Semmelweis University, the National Institute of Oncology and the National Koranyi Institute of TB and Pulmonology. With regards to the molecular factors regulating tumor angiogenesis, we identified the prognostic significance of apelin and erythropoietin receptor (EPOR) expression in non-small cell lung cancer (NSCLC). Furthermore, the impact of KRAS mutation subtypes and ERCC1 (excision repair cross-complementation group 1) expression on the response to platinum-based chemotherapy have been studied. We also described the epidemiology and predictive power of rare EGFR (epidermal growth factor receptor) mutations in a large Hungarian patient cohort. Lastly, the expression of molecular factors associated with NSCLC progression was studied specifically in brain metastatic matched cases series. These preclinical and clinical studies provide clinically relevant information that hopefully will contribute to the improvement of lung cancer patient care.
ABSTRACT
INTRODUCTION: Although classic sensitizing mutations of epidermal growth factor receptor (EGFR) are positive predictive markers for EGFR tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma, there are rare EGFR mutations with unknown epidemiology and influence on prognosis and TKI response. METHODS: Eight hundred and fourteen lung adenocarcinoma patients with KRAS and/or EGFR mutation analyses for TKI therapy indication were identified. Six hundred and forty-five patients were included in the epidemiological analysis. The clinical outcome was analyzed in 419 advanced-stage patients with follow-up data. RESULTS: Four hundred and eighty (59%) KRAS/EGFR double wild-type, 216 (27%) KRAS mutant, 42 (5%) classic, 49 (6%) rare, and 27 (3%) synonymous EGFR mutant cases were identified. Twenty previously unpublished non-synonymous mutations were found. Rare EGFR mutations were significantly associated with smoking (vs. classic EGFR mutations; p = 0.0062). Classic EGFR mutations but not rare ones were independent predictors of increased overall survival (hazard ratios, 0.45; 95% confidence intervals, 0.25-0.82; p = 0.009). TKI therapy response rate of patients harboring classic EGFR mutations was significantly higher (vs. rare EGFR mutations; 71% vs. 37%; p = 0.039). Patients with classic or sensitizing rare (G719x and L861Q) EGFR mutations had significantly longer progression-free survival when compared with the remaining rare mutation cases (12 vs. 6.2 months; p = 0.048). CONCLUSIONS: The majority of rare EGFR mutations was associated with smoking, shorter overall survival, and decreased TKI response when compared with classic EGFR mutations. However, studies characterizing the TKI sensitizing effect of individual rare mutations are indispensable to prevent the exclusion of patients with sensitizing rare EGFR mutations who may benefit from anti-EGFR therapy.
Subject(s)
Adenocarcinoma/epidemiology , Adenocarcinoma/genetics , ErbB Receptors/genetics , Lung Neoplasms/epidemiology , Lung Neoplasms/genetics , Mutation , Adenocarcinoma/drug therapy , Aged , DNA Mutational Analysis , Disease-Free Survival , Female , Humans , Lung Neoplasms/drug therapy , Male , Middle Aged , Molecular Epidemiology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins p21(ras)/genetics , Retrospective Studies , Smoking/epidemiology , Smoking/genetics , Survival RateABSTRACT
BACKGROUND: Platinum-based chemotherapy is the most common treatment in advanced-stage lung adenocarcinoma. Because the clinical significance of KRAS mutational status in this setting has not yet been clearly determined, a mutation subtype-specific analysis was performed in the so far largest cohort of Caucasian patients with KRAS mutant advanced-stage lung adenocarcinoma treated with platinum-based chemotherapy. METHODS: 505 Caucasian stage III-IV lung adenocarcinoma patients with known amino acid substitution-specific KRAS mutational status and treated with platinum-based chemotherapy were included. The correlations of subtype-specific KRAS mutations with smoking status, progression-free and overall survival (PFS and OS, respectively) and therapeutic response were analysed. RESULTS: Among 338 KRAS wild-type, 147 codon 12 mutant and 20 codon 13 mutant patients, there were no mutation-related significant differences in PFS or OS (P values were 0.534 and 0.917, respectively). Eastern Cooperative Oncology Group (ECOG) status and clinical stage were significant independent prognostic factors. KRAS mutation showed a significant correlation with smoking status (P=0.018). Importantly, however, G12V KRAS mutant patients were significantly more frequent among never-smokers than all other codon 12 KRAS mutant (G12x) subtypes (P=0.016). Furthermore, this subgroup tended to have a higher response rate (66% versus 47%; P=0.077). A modestly longer median PFS was also found in the G12V mutant cohort (233days; versus 175days in the G12x group; P=0.145). CONCLUSIONS: While KRAS mutation status per se is neither prognostic nor predictive in stage III-IV lung adenocarcinoma, subtype-specific analysis may indeed identify clinically relevant subgroups of patients that may ultimately influence treatment decisions.