Sensitivity Of Resistant Cells Research Articles

Inhibiting oncogenic signaling by sorafenib activates PUMA via GSK3β and NF-κB to suppress tumor cell growth

Aberrant Ras/Raf/MEK/ERK signaling is one of the most prevalent oncogenic alterations and confers survival advantage to tumor cells. Inhibition of this pathway can effectively suppress tumor cell growth. For example, sorafenib, a multi-kinase inhibitor targeting c-Raf and other oncogenic kinases, has been used clinically for treating advanced liver and kidney tumors, and also has shown efficacy against other malignancies. However, how inhibition of oncogenic signaling by sorafenib and other drugs suppresses tumor cell growth remains unclear. In this study, we found that sorafenib kills cancer cells by activating PUMA, a p53 target and a BH3-only Bcl-2 family protein. Sorafenib treatment induces PUMA in a variety of cancer cells irrespective of their p53 status. Surprisingly, the induction of PUMA by sorafenib is mediated by IκB-independent activation of NF-κB, which directly binds to the PUMA promoter to activate its transcription. NF-κB activation by sorafenib requires GSK3β activation, subsequent to ERK inhibition. Deficiency in PUMA abrogates sorafenib-induced apoptosis and caspase activation, and renders sorafenib resistance in colony formation and xenograft tumor assays. Furthermore, the chemosensitization effect of sorafenib is dependent on PUMA, and involves concurrent PUMA induction through different pathways. BH3 mimetics potentiate the anticancer effects of sorafenib, and restore sorafenib sensitivity in resistant cells. Together, these results demonstrate a key role of PUMA-dependent apoptosis in therapeutic inhibition of Ras/Raf/MEK/ERK signaling. They provide a rationale for manipulating the apoptotic machinery to improve sensitivity and overcome resistance to the therapies that target oncogenic kinase signaling.

Co-Inhibition of BCL-W and BCL2 Restores Antiestrogen Sensitivity through BECN1 and Promotes an Autophagy-Associated Necrosis

BCL2 family members affect cell fate decisions in breast cancer but the role of BCL-W (BCL2L2) is unknown. We now show the integrated roles of the antiapoptotic BCL-W and BCL2 in affecting responsiveness to the antiestrogen ICI 182,780 (ICI; Fulvestrant Faslodex), using both molecular (siRNA; shRNA) and pharmacologic (YC137) approaches in three breast cancer variants; MCF-7/LCC1 (ICI sensitive), MCF-7/LCC9 (ICI resistant), and LY2 (ICI resistant). YC137 inhibits BCL-W and BCL2 and restores ICI sensitivity in resistant cells. Co-inhibition of BCL-W and BCL2 is both necessary and sufficient to restore sensitivity to ICI, and explains mechanistically the action of YC137. These data implicate functional cooperation and/or redundancy in signaling between BCL-W and BCL2, and suggest that broad BCL2 family member inhibitors will have greater therapeutic value than targeting only individual proteins. Whereas ICI sensitive MCF-7/LCC1 cells undergo increased apoptosis in response to ICI following BCL-W±BCL2 co-inhibition, the consequent resensitization of resistant MCF-7/LCC9 and LY2 cells reflects increases in autophagy (LC3 cleavage; p62/SQSTM1 expression) and necrosis but not apoptosis or cell cycle arrest. Thus, de novo sensitive cells and resensitized resistant cells die through different mechanisms. Following BCL-W+BCL2 co-inhibition, suppression of functional autophagy by 3-methyladenine or BECN1 shRNA reduces ICI-induced necrosis but restores the ability of resistant cells to die through apoptosis. These data demonstrate the plasticity of cell fate mechanisms in breast cancer cells in the context of antiestrogen responsiveness. Restoration of ICI sensitivity in resistant cells appears to occur through an increase in autophagy-associated necrosis. BCL-W, BCL2, and BECN1 integrate important functions in determining antiestrogen responsiveness, and the presence of functional autophagy may influence the balance between apoptosis and necrosis.

Abstract A155: Activation of PI3K/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal human malignancies. Sorafenib, a tyrosine kinase inhibitor, was recently approved by FDA for HCC. Material and Methods: We established two sorafenib-resistant HCC cell lines (Huh7-R1 and Huh7-R2) from Huh7, a human HCC cell line, by chronic exposure of cells to sorafenib. The mechanisms of resistance were investigated. Results: Sorafenib induced significant apoptosis in Huh7 cells. However, Huh7-R1 and Huh7-R2 showed significant resistance to sorafenib-induced apoptosis at the clinical relevant concentrations (up to 10 M). Thorough comparisons of the molecular changes between Huh7 and resistant cells, we found PI3K/Akt signaling pathways played a significant role in mediating acquired resistance to sorafenib in Huh7-R1 and Huh7-R2 cells. Our data showed that phospho-Akt and p85 (a regulatory subunit of PI3K) were up-regulated, while PTEN was down-regulated in these resistant cells. In addition, ectopic expression of constitutive Akt in Huh7 demonstrated similar resistance to sorafenib. Knocking down Akt by RNA-interference reversed the resistance to sorafenib in Huh7-R1, indicating the importance of Akt in drug sensitivity. Furthermore, the combination of MK-2206, a novel allosteric Akt inhibitor, and sorafenib restored the sensitivity of resistant cells to sorafenib-induced apoptosis. Conclusions: Activation of PI3K/Akt signaling pathway medicates acquired resistance to sorafenib in HCC cells. The combination of sorafenib with MK-2206, an Akt inhibitor, overcomes the resistance at clinical achievable concentrations. This study is supported by NTUH 98P03, NSC 98-3112-B-002-037, DOH (94–98)-TD-B-111-001 and MK-2206 was kindly provided by Merck & Co., Inc. (Whitehouse Station, NJ, USA). Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A155.

Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death

High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.

ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a γ-secretase inhibitor

ErbB-2 overexpression in breast tumors is associated with poor survival. Expression of Notch-1 and its ligand, Jagged-1, is associated with the poorest survival, including ErbB-2-positive tumors. Trastuzumab plus chemotherapy is the standard of care for ErbB-2-positive breast cancer. A proportion of tumors are initially resistant to trastuzumab and acquired resistance to trastuzumab occurs in metastatic breast cancer and is associated with poor prognosis. Thus, we investigated whether Notch-1 contributes to trastuzumab resistance. ErbB-2-positive cells have low Notch transcriptional activity compared to non-overexpressing cells. Trastuzumab or a dual epidermal growth factor receptor (EGFR)/ErbB-2 tyrosine kinase inhibitor (TKI) increased Notch activity by 2- to 6-fold in SKBr3, BT474 and MCF-7/HER2-18 cells. The increase in activity was abrogated by a Notch inhibitor, gamma-secretase inhibitor (GSI) or Notch-1 small-interfering RNA (siRNA). Trastuzumab decreased Notch-1trade mark precursor, increased amount and nuclear accumulation of active Notch-1(IC) and increased expression of targets, Hey1 and Deltex1 mRNAs, and Hes5, Hey1, Hes1 proteins. Importantly, trastuzumab-resistant BT474 cells treated with trastuzumab for 6 months expressed twofold higher Notch-1, twofold higher Hey1, ninefold higher Deltex1 mRNAs and threefold higher Notch-1 and Hes5 proteins, compared to trastuzumab-sensitive BT474 cells. The increase in Hey1 and Deltex1 mRNAs in resistant cells was abrogated by a Notch-1 siRNA. Cell proliferation was inhibited more effectively by trastuzumab or TKI plus a GSI than either agent alone. Decreased Notch-1 by siRNA increased efficacy of trastuzumab in BT474 sensitive cells and restored sensitivity in resistant cells. Trastuzumab plus a GSI increased apoptosis in sensitive cells by 20-30%. A GSI alone was sufficient to increase apoptosis in trastuzumab-resistant BT474 cells by 20%, which increased to 30% with trastuzumab. Notch-1 siRNA alone decreased cell growth by 30% in sensitive and more than 50% in resistant BT474 cells. Furthermore, growth of both trastuzumab sensitive and resistant cells was completely inhibited by combining trastuzumab plus Notch-1 siRNA. More importantly, Notch-1 siRNA or a GSI resensitized trastuzumab-resistant BT474 cells to trastuzumab. These results demonstrate that ErbB-2 overexpression suppresses Notch-1 activity, which can be reversed by trastuzumab or TKI. These results suggest that Notch-1 might play a novel role in resistance to trastuzumab, which could be prevented or reversed by inhibiting Notch-1.

Defining targets of modulation of human tumor cell response to cisplatin

We previously observed that in yeast cisplatin activates different pathways accounting for stress response. Here, we investigated whether genes involved in yeast drug response were modulated by cisplatin in human tumor cell lines (A2780, IGROV-1, A431, U2-OS) including cisplatin-resistant sublines (A2780/BBR, IGROV-1/Pt1, A431/Pt and U2-OS/Pt). Factors and pathways involved in stress response (glutathione-S-transferase, proteasome, checkpoint control and recombinational repair) were increased by cisplatin in human tumor sensitive and resistant cells. Moreover, sensitization to cisplatin by pharmacologically targeting glutathione or proteasome was observed in sensitive and resistant cells. Interestingly, only in IGROV-1/Pt1 cells, in which cisplatin up-regulated HSP70 and HSP90, targeting of HSP90 resulted in sensitization of resistant cells, suggesting a protective role of stress response. In conclusion, the present findings support the potential relevance of interfering with heat shock protein response to increase cisplatin cytotoxicity in resistant cells. Overall, pathways activated by cisplatin in human tumor cells appear cell-type specific, at least in part reflecting the stress response observed in yeast.

Combined inhibition of mTOR and EGFR with everolimus (RAD001) and gefitinib in patients with non-small cell lung cancer who have smoked cigarettes: A phase II trial

7575 Background: Most non-small cell lung cancers (NSCLC) are primarily resistant to the EGFR tyrosine kinase inhibitor gefitinib. One strategy to overcome resistance is to block parallel or downstream effectors that maintain tumor growth and proliferation. Preclinical data supports this hypothesis as inhibition of the PIK3CA/Akt/mTOR pathway restores gefitinib sensitivity in resistant cells. In a phase I study, gefitinib (250 mg) and the mTOR inhibitor everolimus (5 mg) were safely administered orally once-a-day (Milton Proc ASCO 2005). This combination induced regressions in patients with NSCLC. To assess the efficacy of this regimen, we designed this phase II trial. Methods: Two cohorts of patients with measurable stage IIIB/IV NSCLC were entered: (1) untreated with chemotherapy and (2) previously treated with cisplatin or carboplatin and docetaxel. A Simon two-stage design proposed to enroll up to 31 patients in each cohort. Response was assessed after 1 month then every 2 months. Pretreatment tumor specimens were collected on all patients. Results: To date, 25 patients have entered, 11 untreated and 14 previously treated. The median age was 66 years and KPS 80%. 52% were women, 68% had adenocarcinoma, and all were current (n=1) or former smokers. Partial responses (RECIST) have been observed in 4 of 23 evaluable patients (17%, 95% CI 5 to 39%) One of these 4 tumors harbored a KRAS mutation and one an EGFR exon 19 deletion. Responses lasted 3, 4, 9+, and 16+ months. Responses were observed in 2 of 13 untreated and 2 of 10 previously treated individuals. Toxicities: Grade 2/3 diarrhea in 13%, Grade 2 pustular rash in 25%, and Grade 2 mucosal ulcerations in 38%. 8% had dose reductions. There were no Grade 4 toxicities or treatment-related deaths. Conclusions: The combination of everolimus and gefitinib produced a 17% partial response rate in smokers with NSCLC, a group less likely to benefit from gefitinib. Enough responses have been seen in both untreated and previously treated cohorts to complete enrollment according to the two stage design of this trial. We plan to correlate outcomes with the molecular profile of tumors. Supported by Novartis, USA No significant financial relationships to disclose.

Significant Clinical Activity of the Combination of 5-Azacytidine, Valproic Acid and All-Trans Retinoic (ATRA) Acid in Leukemia: Results of a Phase I/II Study.

5-azacitidine (5-AC), and its analogue 5-aza-2′-deoxycitidine (5-DAC), are DNA hypomethylating agents. Valproic acid (VPA) is a histone deacetylase inhibitor (HDACI). Hypomethylating agents and HDACI restore ATRA sensitivity in resistant cells. The combination of a hypomethylating agent with an HDACI is antileukemic in vitro. Based on this, and on a prior study of 5-DAC and VPA (Garcia-Manero, Blood, in press), we developed a phase I/II study of the combination of 5-AC, VPA and ATRA. Patients (pts) with high risk MDS (≥10% blasts); relapsed/refractory AML; and pts > 60 years with untreated disease and adequate renal, hepatic functions and performance status were eligible. A fixed-dose of 5-AC was used: 75 mg/m2 sq daily x 7. ATRA dose was: 45 mg/m2 PO daily x 5 starting on day 3 of 5-AC. The phase I followed a classic 3+3 design. The initial dose of VPA was 50 mg/kg PO daily x 7 concomitantly with 5-AC. Cycles were at least 21 days long. The phase II targets a response rate of ≥30% with stopping rules. Cohorts of 10 pts (max 40) are studied. Thirty one pts are evaluable. Median age is 60 years (5–78). All, but 2 pts with MDS, had AML. Median number of prior therapies was 2 (0–5). Twenty six pts (83%) had abnormal cytogenetics. During the phase I, at a VPA dose of 50 mg/kg, 1/6 pts developed grade 3 non-hematological toxicity; at 62.5 mg/kg, 2/7, and at 75 mg/kg, 3/6. The dose limiting toxicity (DLT) was neurotoxicity, confusion and somnolence. The MTD was 50 mg/kg daily x 7. Twelve pts are enrolled in the phase II. Of the 31 evaluable pts, 9 achieved CR (ANC 10 9/L, platelets 100 x 10 9/L, and marrow blasts ≤5%) and 3 a CRp (same criteria as of CR but without complete platelet recovery), overall response (OR)39%. The median number of courses to response was 1 (1–3). Nine responses occurred in 16 untreated pts, OR 56% (Table). Eighteen patients were treated at the MTD with 9 responses (50%). Cytogenetic responses were observed in all responding pts. To assess the hypomethylating effect of 5-AC, the LINE test was used. Median LINE methylation pretreatment was 62.5% (57–67%), declined to 58% by day 7 and returned to baseline by day 0 of next cycle (p<0.001). No significant difference was observed between responders and non responders. Eighteen pts had evidence of histone 3 and 4 acetylation (66%). VPA bound level on day 2 was 146 mcg/ml (95% CI 122–170) in responders and 103 mcg/ml (95% CI 88–118) in non-responders, p< 0.005. Analysis of gene re-expression and cell differentiation are ongoing. In conclusion, the combination of 5-AC, VPA and ATRA is safe. The MTD of VPA is 50mg/kg daily x 7. The DLT is neurotoxicity. The combination has significant clinical activity. An OR of 56% was observed in patients > 60 years with previously untreated AML/MDS. Histone acetylation and transient global hypomethylation are observed. Higher levels of VPA are found in responders. Based on prior CALGB experience (Silverman, ASH 2005), this combination appears to be more active than single agent 5-AC in AML. The study continues to accrue.ResponsesVPA (mg/kg)Number of PatientsCRCRpOR%Courses to responseDuration of response (months)501872501(1–3)2.6(0.5–3.2)62.57101423.73+756113318.1(7–9.2+)Total3193381(1–3)2.9(0.5–9.2+)Untreated AML/MDS>60 years1672561(1–3)2.9 (0.5–9.2+)

Phase I/II study of the combination of 5-azacytidine(5-AC), all-trans retinoic acid (ATRA) and valproic Acid (VPA) in patients with myelodysplastic syndrome (MDS) and leukemia

6563 Background: The combination of a hypomethylating agent with a histone deacetylase inhibitor (HDACI) has synergistic activity. The combination of ATRA with either a hypomethylating agent or a HDACI restores ATRA sensitivity in resistant cells. The combination of VPA and ATRA has activity in patients with MDS. Based on this, we developed a phase I/II study of the combination of 5-AC, VPA and ATRA for patients with MDS or AML. Methods: The dose of 5-AC was fixed: 75 mg/m2 sq daily × 7. ATRA dose was: 45 mg/m2 orally daily × 5 starting on day 3. Three dose levels of VPA were studied: 50, 62.5 and 75 mg/kg orally daily × 7. The phase I portion of the study followed a 3+3 design. Patients with high risk MDS (≥10% blasts) or relapsed/refractory AML and patients older than 60 years with untreated disease and adequate renal, hepatic functions and performance status were eligible. Results: Nineteen patients were registered and 16 were evaluable. Median age was 68 years (5–78). All, but one patient with MDS had AML. Median number of prior therapies was 2 (0–5). Twelve patients (75%) had abnormal cytogenetics. At a VPA dose of 50 mg/kg, 1 of 6 patients developed grade 3 confusion; at 62.5 mg/kg, 0 of 6, and at 75 mg/kg, 2 of 6. One patient had a CR, 2 a complete marrow response (marrow blasts &lt; 5%), and 1 a CRP (same criteria as of CR but without complete platelet recovery), overall response rate was 30% of 13 patients that have completed 1 course. All 4 responses occurred during the first cycle. The CR occurred at a VPA dose of 75mg/kg. The other 3 responses occurred at 62.5 and 75 mg/kg. One out of 2 previously untreated older patients achieved a CR. To assess the hypomethylating effect of 5-AC, we used the LINE assay. Median LINE methylation pretreatment was 62.5% (57–67%), declined to 58% by day 7 and returned to baseline by day 0 of next cycle (p&lt;0.001). Analysis of histone acetylation and gene re-expression are ongoing. Conclusions: The combination of 5-AC with VPA and ATRA is well tolerated. The dose of 62.5 mg/kg daily × 7 is currently being expanded. Significant clinical activity was observed and the effects are potentially mediated by the synergistic action of the combination including induction of DNA hypomethylation and histone acetylation. [Table: see text]

Response to histone deacetylase inhibition of novel PML/RARα mutants detected in retinoic acid–resistant APL cells

Resistance to all-trans retinoic acid (ATRA) remains a clinical problem in the treatment of acute promyelocytic leukemia (APL) and provides a model for the development of novel therapies. Molecular alterations in the ligand-binding domain (LBD) of the PML/RARalpha fusion gene that characterizes APL constitute one mechanism of acquired resistance to ATRA. We identified missense mutations in PML/RARalpha from an additional ATRA-resistant patient at relapse and in a novel ATRA-resistant cell line, NB4-MRA1. These cause altered binding to ligand and transcriptional coregulators, leading to a dominant-negative block of transcription. These mutations are in regions of the LBD that appear to be mutational hot spots occurring repeatedly in ATRA-resistant APL patient cells. We evaluated whether histone deacetylase (HDAC) inhibition could overcome the effects of these mutations on ATRA-induced gene expression. Cotreatment with ATRA and TSA restored RARbeta gene expression in NB4-MRA1 cells, whose PML/RARalpha mutation is in helix 12 of the LBD, but not in an APL cell line harboring the patient-derived PML/RARalpha mutation, which was between helix 5 and 6. Furthermore, ATRA combined with TSA increases histone 4 acetylation on the RARbeta promoter only in NB4-MRA1 cells. Consistent with these results, the combined treatment induces differentiation of NB4-MRA1 only. Thus, the ability of an HDAC inhibitor to restore ATRA sensitivity in resistant cells may depend on their specific molecular defects. The variety of PML/RARalpha mutations arising in ATRA-resistant patients begins to explain how APL patients in relapse may differ in response to transcription therapy with HDAC inhibitors.

The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles

Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane. Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent [3H]daunomycin transport which is temperature dependent, osmotically sensitive, and saturable. Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective. ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells. Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation. Only inside-out CMV manifested ATP-dependent daunomycin transport. These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile.

Yeast mutants resistant to basic fuchsin: A genetic approach to the intergration of nuclear and mitochondrial information

In yeast, basic fuchsin both inhibits mitochondrial functions and is a potent mutagen for the mitochondrial genome. A genetic analysis of two mutants resistant to the dye indicate that: (1) resistance is specified by a nuclear gene; (2) the onset of resistance in sensitive cells is controlled by the mitochondiral genome; and (3) sensitive mitochondrial structures can be carried over for many cell generations in the presence of a resistant nuclear genotype.