Extended exposure to low doses of azacitidine induces differentiation of leukemic stem cells through activation of myeloperoxidase.

Oral azacitidine (oral-Aza) treatment results in longer median overall survival (OS) (24.7 vs. 14.8 months in placebo) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy. The dosing schedule of oral-Aza (14 days/28-day cycle) allows for low exposure of Aza for an extended duration thereby facilitating a sustained therapeutic effect. However, the underlying mechanisms supporting the clinical impact of oral-Aza in maintenance therapy remain to be fully understood. In this preclinical work, we explore the mechanistic basis of oral-Aza/extended exposure to Aza through in vitro and in vivo modeling. In cell lines, extended exposure to Aza results in sustained DNMT1 loss, leading to durable hypomethylation, and gene expression changes. In mouse models, extended exposure to Aza, preferentially targets immature leukemic cells. In leukemic stem cell (LSC) models, the extended dose of Aza induces differentiation and depletes CD34+CD38- LSC. Mechanistically, LSC differentiation is driven in part by increased myeloperoxidase (MPO) expression. Inhibition of MPO activity either by using an MPO-specific inhibitor or blocking oxidative stress, a known mechanism of MPO, partly reverses the differentiation of LSC. Overall, our preclinical work reveals novel mechanistic insights into oral-Aza and its ability to target LSC.

Ionotropic receptors mediate olfactory learning and memory in Drosophila.

Phenylacetaldehyde (PAH), an aromatic compound, is present in a diverse range of fruits including overripe bananas and prickly pear cactus, the two major host fruits for Drosophila melanogaster. PAH acts as a potent ligand for the ionotropic receptor 84a (IR84a) in the adult fruit fly and it is detected by the IR84a/IR8a heterotetrameric complex. Its role in the male courtship behavior through IR84a as an environmental aphrodisiac is of additional importance. In D. melanogaster, two distinct kinds of olfactory receptors, that is, odorant receptors (ORs) and ionotropic receptors (IRs), perceive the odorant stimuli. They display unique structural, molecular, and functional characteristics in addition to having different evolutionary origins. Traditionally, olfactory cues detected by the ORs such as ethyl acetate, 1-butanol, isoamyl acetate, 1-octanol, 4-methylcyclohexanol, etc. classified as aliphatic esters and alcohols have been employed in olfactory classical conditioning using fruit flies. This underlines the participation of OR-activated olfactory pathways in learning and memory formation. Our study elucidates that likewise ethyl acetate (EA) (an OR-responsive odorant), PAH (an IR-responsive aromatic compound) too can form learning and memory when associated with an appetitive gustatory reinforcer. The association of PAH with sucrose (PAH/SUC) led to learning and formation of the long-term memory (LTM). Additionally, the Orco1, Ir84aMI00501, and Ir8a1 mutant flies were used to confirm the exclusive participation of the IR84a/IR8a complex in PAH/SUC olfactory associative conditioning. These results highlight the involvement of IRs via an IR-activated pathway in facilitating robust olfactory behavior.

Phenylacetaldehyde induced olfactory conditioning in Drosophila melanogaster (Diptera: Drosophilidae) larvae.

Phenylacetaldehyde (PAH), an aromatic odorant, exists in varied fruits including overripe bananas and prickly pear cactus, the 2 major host fruits of Drosophila melanogaster. It acts as a potent ligand for the Ionotropic receptor 84a (IR84a) and the Odorant receptor 67a (OR67a), serving as an important food and courtship cue for adult fruit flies. Drosophila melanogaster larvae respond robustly to diverse feeding odorants, such as ethyl acetate (EA), an aliphatic ester. Since the chemical identity and concentration of an odorant are vital neural information handled by the olfactory system, we studied how larvae respond to PAH, an aromatic food odorant with aphrodisiac properties for adult flies. Our findings revealed that PAH attracted larvae significantly in a dose-dependent manner. Larvae could also be trained with PAH associated to appetitive and aversive reinforcers. Thus, like EA, PAH might serve as an important odorant cue for larvae, aiding in food tracking and survival in the wild. Since IR84a/IR8a complex primarily governs PAH response in adult flies, we examined expression of Ir84a and Ir8a in early third-instar larvae. Our experiments showed the presence of Ir8a, a novel finding. However, contrary to adult flies, PAH-responsive Ir84a was not found. Our behavioral experiments with Ir8a1 mutant larvae exhibited normal chemotaxis to PAH, whereas Orco1 mutant showed markedly reduced chemotaxis, indicating an OR-mediated neural circuitry for sensing of PAH in larvae. The results obtained through this study are significantly important as information on how larvae perceive and process PAH odorant at the neuronal level is lacking.

Single-cell analysis of localized prostate cancer patients links high Gleason score with an immunosuppressive profile.

BACKGROUND: Evading immune surveillance is a hallmark for the development of multiple cancer types. Whether immune evasion contributes to the pathogenesis of high-grade prostate cancer (HGPCa) remains an area of active inquiry. METHODS: Through single-cell RNA sequencing and multicolor flow cytometry of freshly isolated prostatectomy specimens and matched peripheral blood, we aimed to characterize the tumor immune microenvironment (TME) of localized prostate cancer (PCa), including HGPCa and low-grade prostate cancer (LGPCa). RESULTS: HGPCa are highly infiltrated by exhausted CD8+ T cells, myeloid cells, and regulatory T cells (TRegs). These HGPCa-infiltrating CD8+ T cells expressed high levels of exhaustion markers including TIM3, TOX, TCF7, PD-1, CTLA4, TIGIT, and CXCL13. By contrast, a high ratio of activated CD8+  effector T cells relative to TRegs and myeloid cells infiltrate the TME of LGPCa. HGPCa CD8+  tumor-infiltrating lymphocytes (TILs) expressed more androgen receptor and prostate-specific membran antigen yet less prostate-specific antigen than the LGPCa CD8+  TILs. The PCa TME was infiltrated by macrophages but these did not clearly cluster by M1 and M2 markers. CONCLUSIONS: Our study reveals a suppressive TME with high levels of CD8+ T cell exhaustion in localized PCa, a finding enriched in HGPCa relative to LGPCa. These studies suggest a possible link between the clinical-pathologic risk of PCa and the associated TME. Our results have implications for our understanding of the immunologic mechanisms of PCa pathogenesis and the implementation of immunotherapy for localized PCa.

Gefitinib: An Updated Review of its Role in the Cancer Management, its Nanotechnological Interventions, Recent Patents and Clinical Trials.

BACKGROUND: Gefitinib, a tyrosine kinase inhibitor, is effectively used in the targeted treatment of malignant conditions. It suppresses the signal transduction cascades leading to cell proliferation in the tumors and is now currently approved in several countries globally as secondline and third-line treatment for non-small cell lung cancer (NSCLC). OBJECTIVE: This review is aimed to summarize the journey of gefitinib as an established anticancer drug for the management of various cancers. Moreover, this review will focus on the mechanism of action, established anticancer activities, combination therapy, nanoformulations, as well as recent clinical trials and patents on gefitinib. METHODS: The data for this review was collected from scientific databases such as PubMed, Science Direct, Google Scholar, etc. Recent patents on gefitinib granted in the last two years were collected from databases Patentscope, USPTO, Espacenet, InPASS and Google Patents. Data for the recent clinical trials were obtained from the U.S. National Library of Medicine database. RESULTS: Recent pre-clinical and clinical studies during the period 2015-2021 demonstrating the efficacy of gefitinib were selected and summarized. Total 31 patents were granted in the year 2020-2021 concerning gefitinib. The efficacy of gefitinib against lung cancer, as well as other cancer types, including breast, prostate, colon, cervix etc., was reviewed. CONCLUSION: Gefitinib showed significant advantages in being more effective, safer and more stable, and the associated biopharmaceutical problems are addressed by the application of nanotechnology. The combination therapy using gefitinib and various anticancer molecules of natural and synthetic origin has shown an improved anticancer profile.

Myeloma Genome Project Panel is a Comprehensive Targeted Genomics Panel for Molecular Profiling of Patients with Multiple Myeloma.

PURPOSE: We designed a comprehensive multiple myeloma targeted sequencing panel to identify common genomic abnormalities in a single assay and validated it against known standards. EXPERIMENTAL DESIGN: The panel comprised 228 genes/exons for mutations, 6 regions for translocations, and 56 regions for copy number abnormalities (CNA). Toward panel validation, targeted sequencing was conducted on 233 patient samples and further validated using clinical FISH (translocations), multiplex ligation probe analysis (MLPA; CNAs), whole-genome sequencing (WGS; CNAs, mutations, translocations), or droplet digital PCR (ddPCR) of known standards (mutations). RESULTS: Canonical immunoglobulin heavy chain translocations were detected in 43.2% of patients by sequencing, and aligned with FISH except for 1 patient. CNAs determined by sequencing and MLPA for 22 regions were comparable in 103 samples and concordance between platforms was R2 = 0.969. Variant allele frequency (VAF) for 74 mutations were compared between sequencing and ddPCR with concordance of R2 = 0.9849. CONCLUSIONS: In summary, we have developed a targeted sequencing panel that is as robust or superior to FISH and WGS. This molecular panel is cost-effective, comprehensive, clinically actionable, and can be routinely deployed to assist risk stratification at diagnosis or posttreatment to guide sequencing of therapies.

Reassessment of Therapeutic Applications of Carbon Nanotubes: A Majestic and Futuristic Drug Carrier.

Carbon nanotubes (CNTs) have been identified as one of the most advanced and versatile nanovectors, theranostics, and futuristic drug delivery tools for highly effective delivery of genes, drugs, and biomolecules, as well as for use in bioimaging and as biosensors. CNTs have drawn tremendous attention and interest from researchers worldwide in the past two decades owing to a number of unique characteristics including well defined physicochemical properties, large surface area, in addition to exclusive electrical and optical properties. Numerous recent literature related to the design and applications of CNTs were studied and summarized accordingly. Special emphasis was given for the applications of CNTs in drug targeting. Specific targeting of anticancer drugs such as cisplatin, doxorubicin, taxol, gemcitabine, and methotrexate, and delivery of small interfering RNA, micro-RNA, as well as plasmid DNA have been successfully assisted using CNTs. All the major applications of CNTs were summarized in detail with possible toxicity concerns associated with them. As far as their toxicity is concerned, it was noticed that the functionalized CNTs pose little toxicity and do not have immunogenic effects. In conclusion, CNTs showed great potential in developing a new generation of carriers for various drugs and related biomolecules. The application of CNTs ranges from physics to chemistry and now they are expanding their roles in the therapeutic drug delivery in the modern healthcare system. With applications in every imaginable route of administration, CNTs bring therapeutic benefits to society. The pharmaceutical, biopharmaceutical, pharmacokinetic, pharmacodynamic, and clinical efficacy of CNTs is explored in detail in this review.

Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance.

The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, MS, and superresolution microscopy, we show SMURF2-EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2)-mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF-bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, whereas SMURF2 knockdown decreased EGFR steady-state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR competes with acetylation-mediated receptor internalization that correlates with enhanced receptor stability; therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance.

RIP1 Kinase Drives Macrophage-Mediated Adaptive Immune Tolerance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by immune tolerance and immunotherapeutic resistance. We discovered upregulation of receptor-interacting serine/threonine protein kinase 1 (RIP1) in tumor-associated macrophages (TAMs) in PDA. To study its role in oncogenic progression, we developed a selective small-molecule RIP1 inhibitor with high in vivo exposure. Targeting RIP1 reprogrammed TAMs toward an MHCIIhiTNFα+IFNγ+ immunogenic phenotype in a STAT1-dependent manner. RIP1 inhibition in TAMs resulted in cytotoxic T cell activation and T helper cell differentiation toward a mixed Th1/Th17 phenotype, leading to tumor immunity in mice and in organotypic models of human PDA. Targeting RIP1 synergized with PD1-and inducible co-stimulator-based immunotherapies. Tumor-promoting effects of RIP1 were independent of its co-association with RIP3. Collectively, our work describes RIP1 as a checkpoint kinase governing tumor immunity.

ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation.

Heat shock protein 90 is a chaperone that plays an essential role in the stabilization of a large number of signal transduction molecules, many of which are associated with oncogenesis. An Hsp90 isoform (Hsp90α) has been shown to be selectively phosphorylated on two N-terminal threonine residues (threonine 5 and 7) and is involved in the DNA damage response and apoptosis. However, the kinase that phosphorylates Hsp90α after ionizing radiation (IR) and its role in post-radiation DNA repair remains unclear. Inasmuch as several proteins of the DNA damage response machinery are Hsp90 clients, the functional consequences of Hsp90α phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90α isoform. Here we show that ATM phosphorylates Hsp90α at the T5/7 residues immediately after IR. The kinetics of Hsp90α T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylation (γH2AX). Although Hsp90α is located in both the cytoplasm and nucleus, only nuclear Hsp90α is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90α sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90α null have reduced levels of γH2AX indicating that Hsp90α is important for the formation of γH2AX. Thus, this study provides evidence that Hsp90α is a component of the signal transduction events mediated by ATM following IR, and that Hsp90α loss decreases γH2AX levels. This work supports additional investigation into Hsp90α T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies.

Differential protein stability of EGFR mutants determines responsiveness to tyrosine kinase inhibitors.

Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR-null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance.

Mechanisms of Resistance to EGFR Tyrosine Kinase Inhibitors and Therapeutic Approaches: An Update.

Resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non small cell lung cancer (NSCLC) is mediated by two major mechanisms namely secondary mutation T790M in EGFR and cMET amplification. Other molecular mediators which contribute towards TKI resistance include the activation of compensatory growth signaling, epithelial mesenchymal transition and microRNAs regulating EGFR and cMET levels. In this chapter, we have included the major mechanisms which contribute towards EGFR TKI resistance in NSCLC. Several therapeutic approaches to overcome TKI resistance are also presented which include second and third generation EGFR TKI inhibitors and cMET inhibitors. Further, the rationale to utilize the combination therapies to simultaneously target EGFR and other major oncogene addictive pathway such as ERBB2 and AXL kinase is outlined. Another promising approach to overcome TKI resistance is to potentiate EGFR protein for degradation. These studies will best be utilized when we can identify the oncogene addictions in an individual patient and tailor the therapy/therapies accordingly for the maximum benefits.

Membrane Transition Temperature Determines Cisplatin Response.

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients.

Efficacy of an EGFR-specific peptide against EGFR-dependent cancer cell lines and tumor xenografts.

We have recently synthesized a peptide called Disruptin, which comprised the SVDNPHVC segment of the epidermal growth factor receptor (EGFR) that inhibits binding of heat shock protein 90 (Hsp90) to the EGFR and EGF-dependent EGFR dimerization to cause EGFR degradation. The effect is specific for EGFR versus other Hsp90 client proteins [Ahsan et al.: (2013). Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization. J Biol Chem288, 26879-26886]. Here, we show that Disruptin decreases the clonogenicity of a variety of EGFR-dependent cancer cells in culture but not of EGFR-independent cancer or noncancerous cells. The selectivity of Disruptin toward EGFR-driven cancer cells is due to the high level of EGF stimulation of EGFR in EGFR-dependent tumor cells relative to normal cells. When administered by intraperitoneal injection into nude mice bearing EGFR-driven human tumor xenografts, Disruptin causes extensive degradation of EGFR in the tumor but not in adjacent host tissue. Disruptin markedly inhibits the growth of EGFR-driven tumors without producing the major toxicities caused by the Hsp90 inhibitor geldanamycin or by cisplatin. These findings provide proof of concept for development of a new Disruptin-like class of antitumor drugs that are directed specifically against EGFR-driven tumors.

KRAS protein stability is regulated through SMURF2: UBCH5 complex-mediated ß-TrCP1 degradation.

Attempts to target mutant KRAS have been unsuccessful. Here, we report the identification of Smad ubiquitination regulatory factor 2 (SMURF2) and UBCH5 as a critical E3:E2 complex maintaining KRAS protein stability. Loss of SMURF2 either by small interfering RNA/short hairpin RNA (siRNA/shRNA) or by overexpression of a catalytically inactive mutant causes KRAS degradation, whereas overexpression of wild-type SMURF2 enhances KRAS stability. Importantly, mutant KRAS is more susceptible to SMURF2 loss where protein half-life decreases from >12 hours in control siRNA-treated cells to <3 hours on Smurf2 silencing, whereas only marginal differences were noted for wild-type protein. This loss of mutant KRAS could be rescued by overexpressing a siRNA-resistant wild-type SMURF2. Our data further show that SMURF2 monoubiquitinates UBCH5 at lysine 144 to form an active complex required for efficient degradation of a RAS-family E3, ß-transducing repeat containing protein 1 (ß-TrCP1). Conversely, ß-TrCP1 is accumulated on SMURF2 loss, leading to increased KRAS degradation. Therefore, as expected, ß-TrCP1 knockdown following Smurf2 siRNA treatment rescues mutant KRAS loss. Further, we identify two conserved proline (P) residues in UBCH5 critical for SMURF2 interaction; mutation of either of these P to alanine also destabilizes KRAS. As a proof of principle, we demonstrate that Smurf2 silencing reduces the clonogenic survival in vitro and prolongs tumor latency in vivo in cancer cells including mutant KRAS-driven tumors. Taken together, we show that SMURF2:UBCH5 complex is critical in maintaining KRAS protein stability and propose that targeting such complex may be a unique strategy to degrade mutant KRAS to kill cancer cells.

Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization.

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.

Effect of erlotinib on epidermal growth factor receptor and downstream signaling in oral cavity squamous cell carcinoma.

BACKGROUND: The purpose of this study was to determine if there are differences in biomarker modulation and epidermal growth factor receptor (EGFR) degradation between the tumor and the normal mucosa after treatment with an EGFR inhibitor, erlotinib, in head and neck cancer. METHODS: Patients with primary oral cavity squamous cell cancers received a course of erlotinib, 150 mg every day for 7 days before surgical resection. Tumor and normal mucosa biopsies were obtained both pre-erlotinib and post-erlotinib. Changes in known markers of EGFR activity (phospho, AKT, STAT3) were measured by immunoblotting, whereas changes in tissue distribution were analyzed by immunohistochemical analysis. RESULTS: Twelve patients were enrolled; 7 had evaluable paired tumors and normal mucosa biopsies pretreatment and posttreatment. Expression of EGFR was higher in tumors compared to the normal mucosa (p = .005). Erlotinib administration was associated with marked inhibition of phosphorylated epidermal growth factor receptor (pEGFR) and reduction in total EGFR protein (p = .004, p = .007) in tumors, whereas there was heterogeneity in EGFR inhibition in the normal mucosa (p = .10 [pEGFR], and p = .07 [EGFR]). Reduced levels of pSrc and pSTAT3 and enhanced p27 levels were noted in tumors after erlotinib. Cell culture studies confirmed that EGFR is degraded in tumor cells after prolonged treatment with erlotinib. CONCLUSION: Our results show that EGFR inhibition by erlotinib led to a marked reduction in EGFR protein levels in patients. Differential effects of erlotinib on tumors compared to the normal mucosa suggest there may be individual patient heterogeneity. These preliminary data suggest EGFR degradation should be further analyzed as a potential biomarker in selecting patients likely to benefit from EGFR inhibitors.

Wild-type EGFR is stabilized by direct interaction with HSP90 in cancer cells and tumors.

The epidermal growth factor receptor (EGFR) has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90) is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT) WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA) and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.

Regulation of EGFR protein stability by the HECT-type ubiquitin ligase SMURF2.

Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial tumors and is considered to be an important therapeutic target. Although gene amplification is responsible for EGFR overexpression in certain human malignancies including lung and head and neck cancers, additional molecular mechanisms are likely. Here, we report a novel interaction of EGFR with an HECT-type ubiquitin ligase SMURF2, which can ubiquitinate, but stabilize EGFR by protecting it from c-Cbl-mediated degradation. Conversely, small interfering RNA (siRNA)-mediated knockdown of SMURF2 destabilized EGFR, induced an autophagic response and reduced the clonogenic survival of EGFR-expressing cancer cell lines, with minimal effects on EGFR-negative cancer cells, normal fibroblasts, and normal epithelial cells. UMSCC74B head and neck squamous cancer cells, which form aggressive tumors in nude mice, significantly lost in vivo tumor-forming ability on siRNA-mediated SMURF2 knockdown. Gene expression microarray data from 443 lung adenocarcinoma patients, and tissue microarray data from 67 such patients, showed a strong correlation of expression between EGFR and SMURF2 at the messenger RNA and protein levels, respectively. Our findings suggest that SMURF2-mediated protective ubiquitination of EGFR may be responsible for EGFR overexpression in certain tumors and support targeting SMURF2-EGFR interaction as a novel therapeutic approach in treating EGFR-addicted tumors.