Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity

All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RARα-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RARα activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RARα in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously.

Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data

BACKGROUND The best-known cause of intolerance to fluoropyrimidines is dihydropyrimidine dehydrogenase (DPD) deficiency, which can result from deleterious polymorphisms in the gene encoding DPD (DPYD), including DPYD*2A and c.2846A>T. Three other variants-DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A-have been associated with DPD deficiency, but no definitive evidence for the clinical validity of these variants is available. The primary objective of this systematic review and meta-analysis was to assess the clinical validity of c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity. METHODS We did a systematic review of the literature published before Dec 17, 2014, to identify cohort studies investigating associations between DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A and severe (grade ≥3) fluoropyrimidine-associated toxicity in patients treated with fluoropyrimidines (fluorouracil, capecitabine, or tegafur-uracil as single agents, in combination with other anticancer drugs, or with radiotherapy). Individual patient data were retrieved and analysed in a multivariable analysis to obtain an adjusted relative risk (RR). Effect estimates were pooled by use of a random-effects meta-analysis. The threshold for significance was set at a p value of less than 0·0167 (Bonferroni correction). FINDINGS 7365 patients from eight studies were included in the meta-analysis. DPYD c.1679T>G was significantly associated with fluoropyrimidine-associated toxicity (adjusted RR 4·40, 95% CI 2·08-9·30, p<0·0001), as was c.1236G>A/HapB3 (1·59, 1·29-1·97, p<0·0001). The association between c.1601G>A and fluoropyrimidine-associated toxicity was not significant (adjusted RR 1·52, 95% CI 0·86-2·70, p=0·15). Analysis of individual types of toxicity showed consistent associations of c.1679T>G and c.1236G>A/HapB3 with gastrointestinal toxicity (adjusted RR 5·72, 95% CI 1·40-23·33, p=0·015; and 2·04, 1·49-2·78, p<0·0001, respectively) and haematological toxicity (adjusted RR 9·76, 95% CI 3·03-31·48, p=0·00014; and 2·07, 1·17-3·68, p=0·013, respectively), but not with hand-foot syndrome. DPYD*2A and c.2846A>T were also significantly associated with severe fluoropyrimidine-associated toxicity (adjusted RR 2·85, 95% CI 1·75-4·62, p<0·0001; and 3·02, 2·22-4·10, p<0·0001, respectively). INTERPRETATION DPYD variants c.1679T>G and c.1236G>A/HapB3 are clinically relevant predictors of fluoropyrimidine-associated toxicity. Upfront screening for these variants, in addition to the established variants DPYD*2A and c.2846A>T, is recommended to improve the safety of patients with cancer treated with fluoropyrimidines. FUNDING None.

Preclinical modeling of multi-drug cancer therapies

Anticancer drugs typically are administered in the clinic in the form of mixtures, sometimes called combinations. Only in rare cases, however, are mixtures approved as drugs. Rather, research on mixtures tends to occur after single drugs have been approved. The goal of this research project was to develop modeling approaches that would encourage rational preclinical mixture design. To this end, a series of models were developed. First, several QSAR classification models were constructed to predict the cytotoxicity, oral clearance, and acute systemic toxicity of drugs. The QSAR models were applied to a set of over 115,000 natural compounds in order to identify promising ones for testing in mixtures. Second, an improved method was developed to assess synergistic, antagonistic, and additive effects between drugs in a mixture. This method, dubbed the MixLow method, is similar to the Median-Effect method, the de facto standard for assessing drug interactions. The primary difference between the two is that the MixLow method uses a nonlinear mixed-effects model to estimate parameters of concentration-effect curves, rather than an ordinary least squares procedure. Parameter estimators produced by the MixLow method were more precise than those produced by the Median-Effect Method, and coverage of Loewe index confidence intervals was superior. Third, a model was developed to predict drug interactions based on scores obtained from virtual docking experiments. This represents a novel approach for modeling drug mixtures and was more useful for the data modeled here than competing approaches. The model was applied to cytotoxicity data for 45 mixtures, each composed of up to 10 selected drugs. One drug, doxorubicin, was a standard chemotherapy agent and the others were well-known natural compounds including curcumin, EGCG, quercetin, and rhein. Predictions of synergism/antagonism were made for all possible fixed-ratio mixtures, cytotoxicities of the 10 best-scoring mixtures were tested, and drug interactions were assessed. Predicted and observed responses were highly correlated (r2 = 0.83). Results suggested that some mixtures allowed up to an 11-fold reduction of doxorubicin concentrations without sacrificing efficacy. Taken together, the models developed in this project present a general approach to rational design of mixtures during preclinical drug development. ^

Axl -Gas6 signaling counteracts adenovirus type 5 E1A-mediated cell growth suppression and proapoptotic activity

The adenovirus type 5 E1A (abbreviated E1A) has previously been known as an immortalization oncogene because E1A is required for transforming oncogenes, such as ras and E1B, to transform cells in primary cultures. However, E1A has also been shown to downregulate the overexpression of the Her-2/neu oncogene, resulting in suppression of transformation and tumorigenesis induced by that oncogene. In addition, E1A is able to promote apoptosis induced by anticancer drugs, irradiation, and serum deprivation. Many tyrosine kinases, such as the EGF receptor, Her-2/Neu, Src, and Axl are known to play a role in oncogenic signals in transformed cells. To study the mechanism underlying the E1A-mediated tumor-suppressing function, we exploited a modified tyrosine kinase profile assay (Proc. Natl. Acad. Sci, 93, 5958–5962, 1996) to identify potential tyrosine kinases regulated by E1A. RT-PCR products were synthesized with two degenerate primers derived from the conserved motifs of various tyrosine kinases. A tyrosine kinase downregulated by E1A was identified as Axl by analyzing the Alu I-digested RT-PCR products. We isolated the DNA fragment of interest, and found that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level. To study whether downregulation of the Axl receptor is involved in E1A-mediated growth suppression, we transfected axl cDNA into E1A-expressing cells (ip1-E1A) to establish cells that overexpressed Axl (ip1-E1A-Axl). The Axl ligand Gas6 triggered a greater mitogenic effect in these ip1-E1A-Axl cells than in the control cells ip1-E1A and protected the Axl-expressing cells from serum deprivation-induced apoptosis. Further study showed that Akt is required for Axl-Gas6 signaling to prevent ip1-E1A-Axl cells from serum deprivation-induced apoptosis. These results indicate that downregulation of the Axl receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis, and thereby contributes to E1A's anti-tumor activities. ^

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^

Association of CYP3A4 genotype with treatment-related leukemia

Epipodophyllotoxins are associated with leukemias characterized by translocations of the MLL gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5′ promoter region of the CYP3A4 gene (CYP3A4-V) that might alter the metabolism of anticancer drugs. We examined 99 de novo and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the CYP3A4-V. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99 de novo (19%) and 1 of 30 treatment-related (3%) leukemias carried the CYP3A4-V (P = 0.026; Fisher’s Exact Test, FET). Nine of 42 de novo leukemias with MLL gene translocations (21%), and 0 of 22 treatment-related leukemias with MLL gene translocations carried the CYP3A4-V (P = 0.016, FET). This relationship remained significant when 19 treatment-related leukemias with MLL gene translocations that followed epipodophyllotoxin exposure were compared with the same 42 de novo cases (P = 0.026, FET). These data suggest that individuals with CYP3A4-W genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The CYP3A4-W genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone.

The interferon-inducible murine p48 (ISGF3γ) gene is regulated by protooncogene c-myc

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-γ-regulated element. In addition to the IFN-regulated element, a myc–max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor.

Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death

The correlation between telomerase activity and human tumors has led to the hypothesis that tumor growth requires reactivation of telomerase and that telomerase inhibitors represent a class of chemotherapeutic agents. Herein, we examine the effects of inhibition of telomerase inside human cells. Peptide nucleic acid and 2′-O-MeRNA oligomers inhibit telomerase, leading to progressive telomere shortening and causing immortal human breast epithelial cells to undergo apoptosis with increasing frequency until no cells remain. Telomere shortening is reversible: if inhibitor addition is terminated, telomeres regain their initial lengths. Our results validate telomerase as a target for the discovery of anticancer drugs and supply general insights into the properties that successful agents will require regardless of chemical type. Chemically similar oligonucleotides are in clinical trials and have well characterized pharmacokinetics, making the inhibitors we describe practical lead compounds for testing for an antitelomerase chemotherapeutic strategy.

Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells

Farnesyltransferase inhibitors (FTIs) represent a new class of anticancer drugs that show promise in blocking the growth of tumors. Here, we report that FTIs are capable of inducing apoptosis of transformed but not untransformed cells. Treatment of v-K-ras-transformed normal rat kidney (KNRK) cells with FTIs leads to the induction of apoptotic cell morphology, chromatin condensation and DNA fragmentation. In addition, fluorescence-activated cell sorter analysis of FTI-treated KNRK cells shows a sub-G1 apoptotic peak (chromosome content of <2 N). This FTI-induced apoptosis is evident only when the cells are grown in low serum conditions (0.1% fetal calf serum) and is observed selectively with transformed KNRK cells and not with untransformed NRK cells. Further analysis of the mechanism underlying this apoptosis has shown that FTI treatment of KNRK cells results in the activation of caspase 3 but not caspase 1. Moreover, the addition of Z-DEVD-fmk, an agent that interferes with caspase 3 activity, can inhibit FTI-induced apoptosis in a dose-dependent manner. Introduction of the CASP-3 gene into MCF7 cells, which lack caspase 3 activity, results in a significant increase of FTI-induced apoptosis. Furthermore, FTI induces the release of cytochrome c into the cytosol. This release is an important feature of caspase 3-mediated apoptosis. These results suggest that FTIs induce apoptosis through the release of cytochrome c from the mitochondria resulting in caspase 3 activation.

A multidrug resistance transporter from human MCF-7 breast cancer cells

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 663-aa member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

Eradication of large colon tumor xenografts by targeted delivery of maytansinoids.

The maytansinoid drug DM1 is 100- to 1000-fold more cytotoxic than anticancer drugs that are currently in clinical use. The immunoconjugate C242-DM1 was prepared by conjugating DM1 to the monoclonal antibody C242, which recognizes a mucin-type glycoprotein expressed to various extents by human colorectal cancers. C242-DM1 was found to be highly cytotoxic toward cultured colon cancer cells in an antigen-specific manner and showed remarkable antitumor efficacy in vivo. C242-DM1 cured mice bearing subcutaneous COLO 205 human colon tumor xenografts (tumor size at time of treatment 65-130 mm3), at doses that showed very little toxicity and were well below the maximum tolerated dose. C242-DM1 could even effect complete regressions or cures in animals with large (260- to 500-mm3) COLO 205 tumor xenografts. Further, C242-DM1 induced complete regressions of subcutaneous LoVo and HT-29 colon tumor xenografts that express the target antigen in a heterogeneous manner. C242-DM1 represents a new generation of immunoconjugates that may yet fulfill the promise of effective cancer therapy through antibody targeting of cytotoxic agents.

A standard weight descriptor for dose adjustment in the obese patient

Objective: To develop a standard weight descriptor that can be used for estimation of patient size for obese patients. Patients and methods: Data were available from 3849 patients: 2839 from oncology patients (index data set) and 1010 from general medical patients (validation data set). The patients had a wide range of age (16-100 years), weight (25-165kg) and body mass index (BMI) [12-52 kg/m(2)] in both data sets. From the normal-weight patients in the oncology data set, an equation for male and female patients was developed to predict their normal weight as the sum of the lean body mass and normal fat body mass. The equations were evaluated by predicting the weight of patients in the general medical data set who had a normal BMI (30 kg/m(2)).

Histone deacetylase inhibitors specifically kill nonproliferating tumour cells

Conventional chemotherapeutic drugs target proliferating cells, relying on often small differences in drug sensitivity of tumour cells compared to normal tissue to deliver a therapeutic benefit. Consequently, they have significant limiting toxicities and greatly reduced efficacy against nonproliferating compared to rapidly proliferating tumour cells. This lack of selectivity and inability to kill nonproliferating cells that exist in tumours with a low mitotic index are major failings of these drugs. A relatively new class of anticancer drugs, the histone deacetylase inhibitors (HDI), are selectively cytotoxic, killing tumour and immortalized cells but normal tissue appears resistant. Treatment of tumour cells with these drugs causes both G1 phase cell cycle arrest correlated with increase p21 expression, and cell death, but even the G1 arrested cells died although the onset of death was delayed. We have extended these observations using cells that were stably arrested by either serum starvation or expression of the cyclin-dependent kinase inhibitor p16(ink4a). We report that histone deacetylase inhibitors have similar cytotoxicity towards both proliferating and arrested tumour and immortalized cells, although the onset of apoptosis is delayed by 24 h in the arrested cells. Both proliferating and arrested normal cells are unaffected by HDI treatment. Thus, the histone deacetylase inhibitors are a class of anticancer drugs that have the desirable features of being tumour-selective cytotoxic drugs that are equally effective in killing proliferating and nonproliferating tumour cells and immortalized cells. These drugs have enormous potential for the treatment of not only rapidly proliferating tumours, but tumours with a low mitotic index.

Antisense-mediated inhibition of the plasma membrane Calcium-ATPase suppresses proliferation of MCF-7 cells

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anticancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.