Drug Resistance In Solid Tumors Research Articles

Abstract 2908: EDB+FN is an attractive therapeutic target in oncology: Insights from protein expression analysis of solid tumors

Abstract Stroma plays a major role in the initiation, growth, survival, and drug-resistance of solid tumors, yet few therapeutics specifically target tumor-associated stroma. Extra-domain-B fibronectin (EDB+FN) is an oncofetal splice variant of fibronectin upregulated in solid tumor stroma, which is associated with tumor growth, angiogenesis, and metastases. Here we developed an immunohistochemistry (IHC) assay to assess EDB+FN protein expression in the stroma of tumor and normal tissues to characterize the potential of EDB+FN as a therapeutic target for solid tumors with high unmet need. We validated a primary detection antibody and titrated assay conditions to achieve specificity and a broad dynamic range of EDB+FN expression levels across indications, including thyroid cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, soft tissue sarcoma, pancreatic cancer, non-small cell lung cancer, ovarian cancer, and renal cancer. Considering the stroma-specific expression pattern of EDB+FN, we developed a scoring algorithm weighting both the intensity and distribution of EDB+FN expression specifically in the stroma compartment and separately in tumor cells. Using this assay and scoring approach, we screened more than 200 tumor samples from 10 solid tumor indications and found EDB+FN to be highly differentially expressed in tumor-associated stroma with little to no expression on tumor cells and trace to undetectable levels in healthy tissue. EDB+FN was expressed across the indications evaluated with a wide range of expression both within and between indications. Stromal EDB+FN expression was not correlated with stromal density, which also varied widely within and across indications. In parallel, we developed an algorithm using digital pathology to approximate pathologist scoring of stromal density and EDB+FN expression, allowing streamlined screening of preclinical samples for in vivo studies and indication selection. The disease-specific expression pattern of EDB+FN and high incidence of expression across indications make EDB+FN an ideal target for high-unmet-need solid tumors. The specificity and dynamic range of our IHC assay for detection of EDB+FN protein expression position it as a robust potential biomarker assay for EDB+FN-targeting therapeutics. Citation Format: Sara Lewandowski, Liyang Diao, Alyssa Quigley, Marsha Crochiere, Jan Pinkas. EDB+FN is an attractive therapeutic target in oncology: Insights from protein expression analysis of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2908.

Long non-coding RNAs: controversial roles in drug resistance of solid tumors mediated by autophagy.

Current genome-wide studies have indicated that a great number of long non-coding RNAs (lncRNAs) are transcribed from the human genome and appeared as crucial regulators in a variety of cellular processes. Many studies have displayed a significant function of lncRNAs in the regulation of autophagy. Autophagy is a macromolecular procedure in cells in which intracellular substrates and damaged organelles are broken down and recycled to relieve cell stress resulting from nutritional deprivation, irradiation, hypoxia, and cytotoxic agents. Autophagy can be a double-edged sword and play either a protective or a damaging role in cells depending on its activation status and other cellular situations, and its dysregulation is related to tumorigenesis in various solid tumors. Autophagy induced by various therapies has been shown as a unique mechanism of resistance to anti-cancer drugs. Growing evidence is showing the important role of lncRNAs in modulating drug resistance via the regulation of autophagy in a variety of cancers. The role of lncRNAs in drug resistance of cancers is controversial; they may promote or suppress drug resistance via either activation or inhibition of autophagy. Mechanisms by which lncRNAs regulate autophagy to affect drug resistance are different, mainly mediated by the negative regulation of micro RNAs. In this review, we summarize recent studies that investigated the role of lncRNAs/autophagy axis in drug resistance of different types of solid tumors.

MicroRNA-206 in human cancer: Mechanistic and clinical perspectives

MicroRNAs (miRNAs), small non-coding RNAs approximately 20–25 nt in length, play important roles via directly binding to the corresponding 3′ UTR of target mRNAs. Recent research has shown that miRNAs cover a wide range of diseases, including several types of cancer. It is interesting to note that miR-206 operates as a tumor suppressor and is downregulated in abundant cancer types, such as breast cancer, lung cancer, colorectal cancer, and so forth. Interestingly, a growing number of studies have also reported that miR-206 could function as an oncogene and promote tumor cell proliferation. Thereby, miR-206 may act as either oncogenes or tumor suppressors under certain conditions. In addition, it was widely acknowledged that restoring tumor-suppressor miR-206 has emerged as an unconventional cancer therapy strategy. Therefore, miR-206 might be a newfangled procedure for achieving a more significant treatment outcome for cancer patients. This review summarizes the role of miR-206 in several cancer types and the contributions made between miR-206 and the diagnosis, treatment, and drug resistance of solid tumors.

Analysis of human Tribbles 2 (TRIB2) pseudokinase.

Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a broad human protein interactome, including the well-studied AKT, C/EBPα and MAPK modules. Several lines of evidence indicate that human TRIB2 promotes cell survival and drug-resistance in solid tumors and blood cancers and is therefore of interest as a potential therapeutic target, although its physiological functions remain relatively poorly understood. The unique TRIB2 pseudokinase domain lacks the canonical 'DFG' motif, and subsequently possesses very low affinity for ATP in both the presence and absence of metal ions. However, TRIB2 also contains a unique cysteine-rich αC-helix, which interacts with a conserved peptide motif in its own carboxyl-terminal tail. This regulatory flanking region drives regulated interactions with distinct E3 ubiquitin ligases that serve to control the stability and turnover of TRIB2 client proteins. TRIB2 is also a low-affinity target of several known small-molecule protein kinase inhibitors, which were originally identified using purified recombinant TRIB2 proteins and a thermal shift assay. In this chapter, we discuss laboratory-based procedures for purification, stabilization and analysis of human TRIB2, including screening procedures that can be used for the identification of both reversible and covalent small molecule ligands.

Activatable nanomedicine for overcoming hypoxia-induced resistance to chemotherapy and inhibiting tumor growth by inducing collaborative apoptosis and ferroptosis in solid tumors

Hypoxia has been firmly correlated to the drug resistance of solid tumors. Alleviation of hypoxia by tumor reoxygenation is expected to sensitize the chemotherapy toward solid tumors. Alternatively, ferroptosis provides a therapeutic strategy to overcome apoptotic resistance and multidrug resistance of solid tumors, collaboratively strengthening the chemotherapy toward hypoxic tumors. Herein, an ultrasound (US)-activatable nanomedicine was developed for overcoming hypoxia-induced resistance to chemotherapy and efficiently inhibiting tumor growth by inducing sensitized apoptosis and collaborative ferroptosis of tumor cells. This nanomedicine was constructed by integrating ferrate and doxorubicin into biocompatible hollow mesoporous silica nanoplatforms, followed by assembling a solid-liquid phase-change material of n-heneicosane. The US-induced mild hyperthermia initiates the phase change of n-heneicosane, enabling US-activated co-release of ferrate and doxorubicin. Results reveal that the released ferrate effectively reacts with water as well as the over-expressed hydrogen peroxide and glutathione in tumor cells, achieving tumor-microenvironment-independent reoxygenation and glutathione-depletion in tumors. The reoxygenation down-regulates expressions of hypoxia-inducible factor 1α and multidrug resistance gene/transporter P-glycoprotein in tumor cells, sensitizing the apoptosis-based doxorubicin chemotherapy. More importantly, exogenous iron metabolism from the nanomedicine initiates intracellular Fenton reactions, leading to reactive oxygen species overproduction and iron-dependent ferroptotic death of tumor cells. Furthermore, the glutathione-depletion inactivates the glutathione peroxidase 4 (GPX4, a critical regulatory target in ferroptosis), inhibiting the reduction of lipid peroxides and reinforcing the ferroptotic cell death. The sensitized chemotherapy together with the iron-dependent ferroptosis of tumor cells play a synergistic role in boosting the growth suppression of hypoxic osteosarcoma in vivo. Additionally, the nanomedicine acts as a nanoprobe for in vivo photoacoustic imaging and glutathione tracking, showing great potential as theranostic agents for hypoxic solid tumors treatment.

A Simple Three-Dimensional In Vitro Culture Mimicking the In Vivo-Like Cell Behavior of Bladder Patient-Derived Xenograft Models.

Patient-derived xenograft (PDX) models allow for personalized drug selection and the identification of drug resistance mechanisms in cancer cells. However, PDX models present technical disadvantages, such as long engraftment time, low success rate, and high maintenance cost. On the other hand, tumor spheroids are emerging as an in vitro alternative model that can maintain the phenotype of cancer cells long enough to perform all assays and predict a patient’s outcome. The present work aimed to describe a simple, reproducible, and low-cost 3D in vitro culture method to generate bladder tumor spheroids using human cells from PDX mice. Cancer cells from PDX BL0293 and BL0808 models, previously established from advanced bladder cancer, were cultured in 96-well round-bottom ultra-low attachment (ULA) plates with 5% Matrigel and generated regular and round-shaped spheroids (roundness > 0.8) with a diameter larger than 400 μm and a hypoxic core (a feature related to drug resistance in solid tumors). The responses of the tumor spheroids to the antineoplastic drugs cisplatin, gemcitabine, and their combination were similar to tumor responses in in vivo studies with PDX BL0293 and BL0808 mice. Therefore, the in vitro 3D model using PDX tumor spheroids appears as a valuable tool that may predict the outcome of in vivo drug-screening assays and represents a low-cost strategy for such purpose.

<p>miR-451: A Novel Biomarker and Potential Therapeutic Target for Cancer</p>

MicroRNAs (miRNAs) are endogenous, non-coding, single-stranded small RNAs involved in a variety of cellular processes, including ontogeny, cell proliferation, differentiation, and apoptosis. They can also function as oncogenes or tumor suppressor genes. Recent studies have revealed that miRNA-451 (miR-451) is involved in the regulation of various human physiological and pathological processes. Furthermore, it has been shown that miR-451 not only directly affects the biological functions of tumor cells but also indirectly affects tumor cell invasion and metastasis upon secretion into the tumor microenvironment via exosomes. Thus, miR-451 also influences the progression of tumorigenesis and drug resistance. This review summarizes the expression of miR-451 in various cancer types and the relationship between miR-451 and the diagnosis, treatment, and drug resistance of solid tumors. In addition, we address possible mechanisms of action of miR-451 and its potential application as a biomarker in the diagnosis and treatment of human cancers.

Specific Expression of a New Bruton Tyrosine Kinase Isoform (p65BTK) in the Glioblastoma Gemistocytic Histotype.

Bruton’s tyrosine-kinase (BTK) is a non-receptor tyrosine kinase recently associated with glioma tumorigenesis and a novel prognostic marker for poor survival in patients with glioma. The p65BTK is a novel BTK isoform involved in different pathways of drug resistance of solid tumors, thus we aimed to investigate the expression and the putative role of p65BTK in tumors of the central nervous system (CNS). We selected a large cohort of patients with glial tumors (n = 71) and analyzed the expression of p65BTK in different histotypes and correlation with clinical parameters. Sections were stained with glial fibrillary acidic protein (GFAP), p53, epidermal growth factor receptor (EGFR), S100, vimentin, and epithelial membrane antigen (EMA) antibodies. Glioma stem cell (GSC) lines, isolated from glioblastoma multiforme (GBM), were treated with different concentrations of ibrutinib, a specific inhibitor of BTK, in order to evaluate their metabolic activity, mitotic index and mortality. Moreover, an orthotopic xenotransplant of GSC from human GBM was used to evaluate the expression of p65BTK in the brain of immunodeficient mice. p65BTK was expressed in GSC and in gemistocytes in human gliomas at different histological grade. We found a significant correlation between BTK expression and low-grade (LG) tumors (p ≤ 0.05) and overall survival (OS) of patients with grade III gliomas (p ≤ 0.05), suggestive of worst prognosis. Interestingly, the expression of p65BTK remained restricted exclusively to gemistocytic cells in the xenograft mouse model. Ibrutinib administration significantly reduced metabolic activity and mitotic index and increased mortality in GSC, highlighting the specific role of p65BTK in cell proliferation and survival. In conclusion, our data demonstrated that p65BTK is expressed in glioma tumors, restricted to gemistocytic cells, has a key role in GSC and has a bad prognostic value, thus highlighting the importance of future research for targeted therapy of human gliomas.

CRISPR/Cas9 for overcoming drug resistance in solid tumors.

In this review, we focus on the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated nuclease 9 (Cas9), as a powerful genome editing system, in the identification of resistance mechanisms and in overcoming drug resistance in the most frequent solid tumors. Data were collected by conducting systematic searching of scientific English literature using specific keywords such as "cancer", "CRISPR" and related combinations. The review findings revealed the importance of CRISPR/Cas9 system in understanding drug resistance mechanisms and identification of resistance-related genes such as PBRM1, SLFN11 and ATPE1 in different cancers. We also provided an overview of genes, including RSF1, CDK5, and SGOL1, whose disruption can synergize with the currently available drugs such as paclitaxel and sorafenib. The data suggest CRISPR/Cas9 system as a useful tool in elucidating the molecular basis of drug resistance and improving clinical outcomes. Graphical abstract The mechanisms of CRISPR/Cas9-mediated genome editing and double-strand breaks (DSBs) repair.

PO-278 The Mechanism Of LIL combined with AZD8055 On The Glutamine Addiction Of CT26 Cells

Objective The photobiomodulation of low-intensity lasers can activate many pathways and molecules, which involved in tumor growth, invasion, metastasis, and resistance to drugs or radiation therapy, but studies of low-intensity lasers on tumor cell addiction, have not been reported. Numerous studies have shown that changes in plasma and muscle glutamine levels can occur when the body undergoes prolonged exercise (>1 hour) or overtraining. AZD8055 is a dual inhibitor of mammalian rapamycin complex (mTORC)1/mTORC2 that inhibits phosphorylation of mTORC1 (p70S6K and 4E-BP1), mTORC2 (AKT) and downstream proteins.
 Based on the experimental model of glutamine addiction in laboratory, we chose the worst conditions to cause super-proliferation of CT26 cells and simulate the drug resistance of solid tumors. The purpose is to study tumor deterioration from the perspective of tumor cell function. Under the state, the effect of low-intensity laser combined with AZD8055 on the proliferation of glutamine addiction in CT26 cells is also the development of exercise intervention tumors, and the experimental basis is proposed from the perspective of nutrient metabolism.
 Methods This experiment used a pre-experimental glutamine addiction model to culture mouse colon cancer CT26 cells after horse serum shock, and used different intensity 640±15 nm low-intensity lasers from light-emitting diode arrays (red light at 640±15 nm from Light emitting diode array, RLED), 15 min/day；different concentrations of AZD8055 (0 ~ 100μM) on different proliferative CT26 cells at different times.
 Cell proliferation was detected by MTT assay; mRNA levels of proliferation-related genes were detected by RT-PCR; protein levels were detected by western blot.
 The data were analyzed by SPSS21.0 statistical software and self-similarity index analysis. Among them, the statistical significance level was set at 0.05, P<0.05 was different; P<0.01 was significant difference. In the self-similarity index analysis method, l>0.8 is a significant difference.
 Results 1.CT26 cells were incubated at Gln7.94 mM until the 8th day, and AZD8055 was added at different concentrations. Compared with no inhibitor , and at 6 h, the cells were significantly increased when the concentration of AZD8055 was 0.001 μM, 1 μM, 10 μM and 100 μM. (P<0.01); at 12h, the cells were significantly inhibition when the of AZD8055>0.5μM (P<0.01).
 2.From the growth inhibition rate analysis, at 48 h and 72 h, the inhibition rate exceeded 50%,when the concentration of AZD8055 was 100 μM (61.01% and 87.46%), when< 0.1 μM, the cells were proliferation occurred at 48 h and 72 h, and it is drug resistance.
 3.When AZD8055 combined with low-intensity laser treatment of CT26 cells, CT26 cells had different degrees of proliferative effects at different time points: 12h, 24h, 48h and 72h. Especially at 48h, the light intensity of LIL2 to LIL8 had a significant proliferative effect on CT26 cells (l≥0.8).
 4.Compared with the control group (0 hours without inhibitor), the CT26 cells were treatment with AZD8055 1μM , the mRNA level of the gene GLUT1 was significantly down-regulated in all time periods (P<0.01). When the LIL(2.17 mW/cm2) combined AZD8055 (1μM) significantly promoted the proliferation of CT26 cells at 24h and 48h. It is related to up-regulated of protein expression in all time points: at 6h and 12h, the pMEK protein and the pMEK/ MEK were significantly up-regulated(P<0.01). At 24h, the expression of GLUT1 protein was up-regulated(P<0.01), MEK protein, pERK protein and ERK/ pERK were significantly up-regulated (P<0.01). At 48h, the GLUT1 protein, MEK protein , pERK protein and pERK/ERK were up-regulated (P<0.01).
 Conclusions 1.The minimum inhibitory concentration of AZD8055 on super-proliferative CT26 cells was 0.5 μM, and the super-proliferative state showed more obvious drug resistance.
 2.When low-intensity lasers combined with AZD8055 treated CT26 super-proliferating cells, CT26 cells are not sensitive to AZD8055, while low-intensity lasers promote CT26 cells proliferation by up-regulating MEK/ERK signaling pathways.

Modeling the chemotherapy-induced selection of drug-resistant traits during tumor growth

The emergence of drug-resistance is a major challenge in chemotherapy. In this paper we develop a mathematical model to study the dynamics of drug-resistance in solid tumors. Our model follows the dynamics of the tumor, assuming that the cancer cell population depends on a phenotype variable that corresponds to the resistance level to a cytotoxic drug. The equation for the tumor density is written as a reaction-diffusion equation with a pressure term that depends on the local cell density. The model incorporates the dynamics of nutrients and two different types of drugs: a cytotoxic drug, which directly impacts the death rate of the cancer cells, and a cytostatic drug that reduces the proliferation rate. This model successfully integrates the phenotype structured drug-resistance approach with an asymmetric tumor growth model in space. Through analysis and simulations we study the impact of spatial and phenotypic heterogeneity on the tumor growth under chemotherapy. We demonstrate that heterogeneous cancer cells may emerge due to the selection dynamics of the environment. Our model predicts that under certain conditions, multiple resistant traits emerge at different locations within the tumor. We show that a higher dosage of the cytotoxic drug may delay a relapse, yet, when this happens, a more resistant trait emerges. Moreover, we estimate the expansion rate of the tumor boundary as well as the time of relapse, in terms of the resistance trait, the level of the nutrient, and the drug concentration. Finally, we propose an efficient drug schedule aiming at minimizing the growth rate of the most resistant trait. By combining the cytotoxic and cytostatic drugs, we demonstrate that the resistant cells can be eliminated.

Modeling the Transfer of Drug Resistance in Solid Tumors.

ABC efflux transporters are a key factor leading to multidrug resistance in cancer. Overexpression of these transporters significantly decreases the efficacy of anti-cancer drugs. Along with selection and induction, drug resistance may be transferred between cells, which is the focus of this paper. Specifically, we consider the intercellular transfer of P-glycoprotein (P-gp), a well-known ABC transporter that was shown to confer resistance to many common chemotherapeutic drugs. In a recent paper, Durán et al. (Bull Math Biol 78(6):1218-1237, 2016) studied the dynamics of mixed cultures of resistant and sensitive NCI-H460 (human non-small lung cancer) cell lines. As expected, the experimental data showed a gradual increase in the percentage of resistance cells and a decrease in the percentage of sensitive cells. The experimental work was accompanied with a mathematical model that assumed P-gp transfer from resistant cells to sensitive cells, rendering them temporarily resistant. The mathematical model provided a reasonable fit to the experimental data. In this paper, we develop a new mathematical model for the transfer of drug resistance between cancer cells. Our model is based on incorporating a resistance phenotype into a model of cancer growth (Greene etal. in J Theor Biol 367:262-277, 2015). The resulting model for P-gp transfer, written as a system of integro-differential equations, follows the dynamics of proliferating, quiescent, and apoptotic cells, with a varying resistance phenotype. We show that this model provides a good match to the dynamics of the experimental data of Durán etal. (2016). The mathematical model shows a better fit when resistant cancer cells have a slower division rate than the sensitive cells.

Co-delivery of oxygen and erlotinib by aptamer-modified liposomal complexes to reverse hypoxia-induced drug resistance in lung cancer.

Tumor hypoxia is a common feature of the tumor microenvironment and has been regarded as one of the key factors in driving the emergence of drug resistance in solid tumors. To surmount the hypoxia-associated drug resistance, we fabricated the novel multifunctional liposomal complexes (ACLEP) that could co-deliver oxygen and molecular targeted drug to overcome the hypoxia-induced drug resistance in lung cancer. The ACLEP were fabricated with liposomes anchored with anti-EGFR aptamer-conjugated chitosan to co-administrate erlotinib and PFOB to EGFR-overexpressing non-small-cell lung cancer. Our results showed that the ACLEP possessed desired physicochemistry, good biostability and controlled drug release. The entrapped PFOB in nanoparticle facilitated the uptake of ACLEP in either normoxia or hypoxic condition. Comparing to those nanoparticles loading erlotinib alone, our innovative oxygen/therapeutic co-delivery system showed a promising outcome in fighting against hypoxia-evoked erotinib resistance both invitro and invivo. Hence, this work presents a potent drug delivery platform to overcome hypoxia-induced chemotherapy resistance.

Abstract 3887: GNA13 is a theranostic target that drives drug resistance and cancer stem-like phenotypes in solid tumors

Abstract Treatment failure in solid tumors occurs due to the survival of specific subpopulations of cells that possess stem cell-like (CSC) phenotypes. Studies have implicated G protein-coupled-receptors (GPCRs) in cancer progression and the acquisition of aggressive phenotypes. Many of the implicated GPCRs signal through the G12 subfamily, comprised of GNA12 and GNA13. In this study, we demonstrate that GNA13 is upregulated in many solid tumors and impacts survival and metastases in these patients. Consistent with this, we show that GNA13 expression modulates drug resistance through its effect on the CSC sub-population in a panel of patient-derived head and neck (HNSCC) and breast cancer cells. These data were validated in vivo, where GNA13 over-expression in patient-derived xenografts increased tumor initiating capacity, tumorigenicity and drug resistance, with no effect on growth or proliferation. Signaling through NFKB and MAPK pathways appear to be critical to the observed phenotype. Importantly, blockade of GNA13 expression, or select downstream pathways using small-molecule inhibitors, abrogates GNA13-induced CSCs, rendering cells vulnerable to standard-of-care cytotoxic therapy for these cancers. Taken together, these data indicate that GNA13 expression is a potential prognostic biomarker, and interfering with GNA13-induced signaling provides a novel strategy to block CSCs and drug resistance in solid tumors. Citation Format: Suhail Ahmed Kabeer Rasheed, Hui Sun Leong, Manikandan Lakshmanan, Anandhkumar Raju, Dhivya Dadlani, Fui-Teen Chong, Ravisankar Rajarethinam, Thakshayeni Skanthakumar, Ern Yu Tan, Jacqueline Siok Gek Hwang, Kok Hing Lim, Daniel Shao-Weng Tan, Paolo Ceppi, Mei Wang, Vinay Tergaonkar, Patrick J. Casey, N. Gopalakrishna Iyer. GNA13 is a theranostic target that drives drug resistance and cancer stem-like phenotypes in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3887. doi:10.1158/1538-7445.AM2017-3887

Transforming doxorubicin into a cancer stem cell killer via EpCAM aptamer-mediated delivery.

Chemotherapy-resistant cancer stem cells (CSCs) are a major obstacle to the effective treatment of many forms of cancer. To overcome CSC chemo-resistance, we developed a novel system by conjugating a CSC-targeting EpCAM aptamer with doxorubicin (Apt-DOX) to eliminate CSCs. Incubation of Apt-DOX with colorectal cancer cells resulted in high concentration and prolonged retention of DOX in the nuclei. Treatment of tumour-bearing xenograft mice with Apt-DOX resulted in at least 3-fold more inhibition of tumour growth and longer survival as well as a 30-fold lower frequency of CSC and a prolonged longer tumourigenic latency compared with those receiving the same dose of free DOX. Our data demonstrate that a CSC-targeting aptamer is able to transform a conventional chemotherapeutic agent into a CSC-killer to overcome drug resistance in solid tumours.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Leveraging Hypoxia-Activated Prodrugs to Prevent Drug Resistance in Solid Tumors.

Experimental studies have shown that one key factor in driving the emergence of drug resistance in solid tumors is tumor hypoxia, which leads to the formation of localized environmental niches where drug-resistant cell populations can evolve and survive. Hypoxia-activated prodrugs (HAPs) are compounds designed to penetrate to hypoxic regions of a tumor and release cytotoxic or cytostatic agents; several of these HAPs are currently in clinical trial. However, preliminary results have not shown a survival benefit in several of these trials. We hypothesize that the efficacy of treatments involving these prodrugs depends heavily on identifying the correct treatment schedule, and that mathematical modeling can be used to help design potential therapeutic strategies combining HAPs with standard therapies to achieve long-term tumor control or eradication. We develop this framework in the specific context of EGFR-driven non-small cell lung cancer, which is commonly treated with the tyrosine kinase inhibitor erlotinib. We develop a stochastic mathematical model, parametrized using clinical and experimental data, to explore a spectrum of treatment regimens combining a HAP, evofosfamide, with erlotinib. We design combination toxicity constraint models and optimize treatment strategies over the space of tolerated schedules to identify specific combination schedules that lead to optimal tumor control. We find that (i) combining these therapies delays resistance longer than any monotherapy schedule with either evofosfamide or erlotinib alone, (ii) sequentially alternating single doses of each drug leads to minimal tumor burden and maximal reduction in probability of developing resistance, and (iii) strategies minimizing the length of time after an evofosfamide dose and before erlotinib confer further benefits in reduction of tumor burden. These results provide insights into how hypoxia-activated prodrugs may be used to enhance therapeutic effectiveness in the clinic.

JAK-mediated autophagy regulates stemness and cell survival in cisplatin resistant bladder cancer cells

BackgroundAutophagy is a critical process in acquiring drug resistance in solid tumors. However, the mechanisms by which autophagy modulate resistance to chemotherapy in bladder cancer remains poorly understood. Material and methodsWe have established cisplatin resistant patient derived primary cultured cells as well as T24 bladder cancer cells. The autophagy flux as well as the effect of chemotherapeutic agents, gemcitabine (GC) and mitomycin (MM) were evaluated in these cells. Side population analysis, tumorisphere assay and expression of stemness genes were also studied to evaluate the role of autophagy during chemoresistance. The role of cytokines in autophagy induced chemoresistance was demonstrated. ResultsOur results show that resistant cells have higher basal autophagic flux. GC and MM led to increase in autophagy and combination of autophagy inhibitors synergistically inhibited bladder cancer cell growth. When autophagy was inhibited, only IFN-γ level was decreased in cytokine array. Blocking IFN-γ decreased JAK2 expression and reduced the autophagy. Inhibition of JAK2 decreased the proportion of cancer stem cell enriched side population, tumor sphere forming ability and led to decrease in expression of stemness genes. Combined treatment with inhibitors of JAK2 and autophagy led to inhibition in cell growth, reduced the levels of inflammatory cytokines and decreased the resistance gene expression. ConclusionGC and MM treatment increased autophagy process via IFN-γ mediated JAK2 and STAT3 pathway. General significanceThese findings support a key role of the autophagy in drug resistance. Thus, autophagy-based, “customized” combinatorial approaches may effectively manage resistance and relapse in bladder cancer.

Abstract 4698: BET protein inhibitors prevent TKI-induced drug resistance in solid tumors

Abstract Acquired drug resistance is one of the major challenges for clinical application of tyrosine kinase inhibitor (TKI) in cancer treatment. TKI-induced drug resistance could develop as a result of adaptive cell reprogramming that leads to pathway feedback activation in the same signaling pathway or parallel pathways or gatekeeper mutations abolishing drug binding. The findings of reversible drug response and lack of genetic mutations in TKI-resistant tumor cells suggest epigenetic basis during resistance development process. Targeting epigenetic modifiers and readers, in particular, the bromodomain and extra-terminal (BET) protein family, has shown promising efficacies against both primary and drug-resistant hematological malignancies via modulating transcription profile and suppressing the MYC oncogene. BGB-3619 is a novel, potent inhibitor of BET family proteins (BRD2, BRD3, BRD4 and BRDT) with IC50 ranging from 20 to 49 nM. It demonstrates superior anti-proliferation activity than iBET762 in multiple leukemia and lymphoma cell lines. In this study, we investigated the effect of BGB-3619 and iBET762 on solid tumor cells during and after TKI-resistance development. From cell killing assays, BET inhibitors were found to be much more potent to TKI-resistant cells (vemurafenib-resistant A375, HT29, SK-MEL-5, cells and erlotinib-resistant HCC827 cells) than their parental cells. Notably, long-term colony formation assays showed that combination of low dose BET inhibitors and TKI effectively suppressed the expansion of TKI-tolerated cell populations. The enhanced sensitivity of TKI-resistant cells to BET inhibitor was due to strong G1 phase arrest rather than induction of apoptosis. Furthermore, BET inhibitors blocked cell proliferation independent of c-Myc inhibition, suggesting they might affect a broad transcription network and provide favorable epigenetic state to induce cell cycle arrest. Taken together, these findings suggested potential therapeutic activity of BET inhibitor in TKI-resistant solid tumors and a strategy of combining BET inhibitors with TKIs to overcome resistance to targeted cancer therapies. Citation Format: Yuan Zhao, Yiyuan Wu, Hexiang Wang, Bo Ren, Ye Liu, Min Wei, Changyou Zhou, Lusong Luo. BET protein inhibitors prevent TKI-induced drug resistance in solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4698.

Modeling the effects of space structure and combination therapies on phenotypic heterogeneity and drug resistance in solid tumors.

Histopathological evidence supports the idea that the emergence of phenotypic heterogeneity and resistance to cytotoxic drugs can be considered as a process of selection in tumor cell populations. In this framework, can we explain intra-tumor heterogeneity in terms of selection driven by the local cell environment? Can we overcome the emergence of resistance and favor the eradication of cancer cells by using combination therapies? Bearing these questions in mind, we develop a model describing cell dynamics inside a tumor spheroid under the effects of cytotoxic and cytostatic drugs. Cancer cells are assumed to be structured as a population by two real variables standing for space position and the expression level of a phenotype of resistance to cytotoxic drugs. The model takes explicitly into account the dynamics of resources and anticancer drugs as well as their interactions with the cell population under treatment. We analyze the effects of space structure and combination therapies on phenotypic heterogeneity and chemotherapeutic resistance. Furthermore, we study the efficacy of combined therapy protocols based on constant infusion and bang-bang delivery of cytotoxic and cytostatic drugs.