EGFR in Cancer: Signaling Mechanisms, Drugs, and Acquired Resistance.

The epidermal growth factor receptor (EGFR) has served as the founding member of the large family of growth factor receptors harboring intrinsic tyrosine kinase function. High abundance of EGFR and large internal deletions are frequently observed in brain tumors, whereas point mutations and small insertions within the kinase domain are common in lung cancer. For these reasons EGFR and its preferred heterodimer partner, HER2/ERBB2, became popular targets of anti-cancer therapies. Nevertheless, EGFR research keeps revealing unexpected observations, which are reviewed herein. Once activated by a ligand, EGFR initiates a time-dependent series of molecular switches comprising downregulation of a large cohort of microRNAs, up-regulation of newly synthesized mRNAs, and covalent protein modifications, collectively controlling phenotype-determining genes. In addition to microRNAs, long non-coding RNAs and circular RNAs play critical roles in EGFR signaling. Along with driver mutations, EGFR drives metastasis in many ways. Paracrine loops comprising tumor and stromal cells enable EGFR to fuel invasion across tissue barriers, survival of clusters of circulating tumor cells, as well as colonization of distant organs. We conclude by listing all clinically approved anti-cancer drugs targeting either EGFR or HER2. Because emergence of drug resistance is nearly inevitable, we discuss the major evasion mechanisms.

TSHZ2 is an EGF-regulated tumor suppressor that binds to the cytokinesis regulator PRC1 and inhibits metastasis.

Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.

Retinal Proteomics of a Mouse Model of Dystroglycanopathies Reveals Molecular Alterations in Photoreceptors.

Mutations in the POMT1 gene, encoding a protein O-mannosyltransferase essential for α-dystroglycan (α-DG) glycosylation, are frequently observed in a group of rare congenital muscular dystrophies, collectively known as dystroglycanopathies. However, it is hitherto unclear whether the effects seen in affected patients can be fully ascribed to α-DG hypoglycosylation. To study this, here we used comparative mass spectrometry-based proteomics and immunofluorescence microscopy and investigated the changes in the retina of mice in which Pomt1 is specifically knocked out in photoreceptor cells. Our results demonstrate significant proteomic changes and associated structural alteration in photoreceptor cells of Pomt1 cKO mice. In addition to the effects related to impaired α-DG O-mannosylation, we observed morphological alterations in the outer segment that are associated with dysregulation of a relatively understudied POMT1 substrate (KIAA1549), BBSome proteins, and retinal stress markers. In conclusion, our study provides new hypotheses to explain the phenotypic changes that are observed in the retina of patients with dystroglycanopathies.

Host-Dependent Phenotypic Resistance to EGFR Tyrosine Kinase Inhibitors.

Lung cancers driven by mutant forms of EGFR invariably develop resistance to kinase inhibitors, often due to secondary mutations. Here we describe an unconventional mechanism of resistance to dacomitinib, a newly approved covalent EGFR kinase inhibitor, and uncover a previously unknown step of resistance acquisition. Dacomitinib-resistant (DR) derivatives of lung cancer cells were established by means of gradually increasing dacomitinib concentrations. These DR cells acquired no secondary mutations in the kinase or other domains of EGFR. Along with resistance to other EGFR inhibitors, DR cells acquired features characteristic to epithelial-mesenchymal transition, including an expanded population of aldehyde dehydrogenase-positive cells and upregulation of AXL, a receptor previously implicated in drug resistance. Unexpectedly, when implanted in animals, DR cells reverted to a dacomitinib-sensitive state. Nevertheless, cell lines derived from regressing tumors displayed renewed resistance when cultured in vitro. Three-dimensional and cocultures along with additional analyses indicated lack of involvement of hypoxia, fibroblasts, and immune cells in phenotype reversal, implying that other host-dependent mechanisms might nullify nonmutational modes of resistance. Thus, similar to the phenotypic resistance of bacteria treated with antibiotics, the reversible resisters described here likely evolve from drug-tolerant persisters and give rise to the irreversible, secondary mutation-driven nonreversible resister state. SIGNIFICANCE: This study reports that stepwise acquisition of kinase inhibitor resistance in lung cancers driven by mutant EGFR comprises a nonmutational, reversible resister state. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/14/3862/F1.large.jpg.

Upfront admixing antibodies and EGFR inhibitors preempts sequential treatments in lung cancer models.

Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anti-cancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first-, second-, and third-generation drugs. To explore alternatives to such whack-a-mole strategies, we simulated in patient-derived xenograft models the situation of patients receiving first-line TKIs. Monotherapies comprising approved first-line TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drug-drug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistance-conferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing next-generation TKIs.

A role for DJ-1 against oxidative stress in the mammalian retina.

We have previously reported the expression of Parkinson disease-associated genes encoding α-synuclein, parkin and UCH-L1 in the retina across mammals. DJ-1, or parkinsonism-associated deglycase, is a redox-sensitive protein with putative roles in cellular protection against oxidative stress, among a variety of functions, acting through distinct pathways and mechanisms in a wide variety of tissues. Its function in counteracting oxidative stress in the retina, as it occurs in Parkinson and other human neurodegenerative diseases, is, however, poorly understood. In the present study, we address the expression of DJ-1 in the mammalian retina and its putative neuroprotective role in this tissue in a well-known model of parkinsonism, the rotenone-treated rat. As a result, we demonstrate that the DJ1 gene is expressed at both mRNA and protein levels in the neural retina and retinal pigment epithelium (RPE) of all mammalian species studied. We also present evidence that DJ-1 functions in the retina as a sensor of cellular redox homeostasis, which reacts to oxidative stress by increasing its intracellular levels and additionally becoming oxidized. Levels of α-synuclein also became upregulated, although parkin and UCH-L1 expression remained unchanged. It is inferred that DJ-1 likely exerts in the retina a potential neuroprotective role against oxidative stress, including α-synuclein oxidation and aggregation, which should be operative under both physiological and pathological conditions.

Impairment of photoreceptor ribbon synapses in a novel Pomt1 conditional knockout mouse model of dystroglycanopathy.

Hypoglycosylation of α-dystroglycan (α-DG) resulting from deficiency of protein O-mannosyltransferase 1 (POMT1) may cause severe neuromuscular dystrophies with brain and eye anomalies, named dystroglycanopathies. The retinal involvement of these disorders motivated us to generate a conditional knockout (cKO) mouse experiencing a Pomt1 intragenic deletion (exons 3-4) during the development of photoreceptors, mediated by the Cre recombinase expressed from the cone-rod homeobox (Crx) gene promoter. In this mouse, retinal α-DG was unglycosylated and incapable of binding laminin. Retinal POMT1 deficiency caused significant impairments in both electroretinographic recordings and optokinetic reflex in Pomt1 cKO mice, and immunohistochemical analyses revealed the absence of ß-DG and of the α-DG-interacting protein, pikachurin, in the outer plexiform layer (OPL). At the ultrastructural level, noticeable alterations were observed in the ribbon synapses established between photoreceptors and bipolar cells. Therefore, O-mannosylation of α-DG in the retina carried out by POMT1 is crucial for the establishment of proper synapses at the OPL and transmission of visual information from cones and rods to their postsynaptic neurons.

Expression in retinal neurons of fukutin and FKRP, the protein products of two dystroglycanopathy-causative genes.

Purpose: Dystroglycanopathies are a heterogeneous group of recessive neuromuscular dystrophies that affect the muscle, brain and retina, and are caused by deficiencies in the O-glycosylation of α-dystroglycan. This post-translational modification is essential for the formation and maintenance of ribbon synapses in the retina. Fukutin and fukutin-related protein (FKRP) are two glycosyltransferases whose deficiency is associated with severe dystroglycanopathies. These enzymes carry out in vitro the addition of a tandem ribitol 5-phosphate moiety to the so-called core M3 phosphotrisaccharide of α-dystroglycan. However, their expression pattern and function in the healthy mammalian retina has not so far been investigated. In this work, we have addressed the expression of the FKTN (fukutin) and FKRP genes in the retina of mammals, and characterized the distribution pattern of their protein products in the adult mouse retina and the 661W photoreceptor cell line. Methods: By means of reverse transcription (RT)-PCR and immunoblotting, we have studied the expression at the mRNA and protein levels of the fukutin and FKRP genes in different mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of their protein products in mouse retinal sections and in 661W cultured cells. Results: Both genes were expressed at the mRNA and protein levels in the neural retina of all mammals studied. Fukutin was present in the cytoplasmic and nuclear fractions in the mouse retina and 661W cells, and accumulated in the endoplasmic reticulum. FKRP was located in the cytoplasmic fraction in the mouse retina and concentrated in the Golgi complex. However, and in contrast to retinal tissue, FKRP additionally accumulated in the nucleus of the 661W photoreceptors. Conclusions: Our results suggest that fukutin and FKRP not only participate in the synthesis of O-mannosyl glycans added to α-dystroglycan in the endoplasmic reticulum and Golgi complex, but that they could also play a role, that remains to be established, in the nucleus of retinal neurons.

Expression pattern in retinal photoreceptors of POMGnT1, a protein involved in muscle-eye-brain disease.

PURPOSE: The POMGNT1 gene, encoding protein O-linked-mannose ß-1,2-N-acetylglucosaminyltransferase 1, is associated with muscle-eye-brain disease (MEB) and other dystroglycanopathies. This gene's lack of function or expression causes hypoglycosylation of α-dystroglycan (α-DG) in the muscle and the central nervous system, including the brain and the retina. The ocular symptoms of patients with MEB include retinal degeneration and detachment, glaucoma, and abnormal electroretinogram. Nevertheless, the POMGnT1 expression pattern in the healthy mammalian retina has not yet been investigated. In this work, we address the expression of the POMGNT1 gene in the healthy retina of a variety of mammals and characterize the distribution pattern of this gene in the adult mouse retina and the 661W photoreceptor cell line. METHODS: Using reverse transcription (RT)-PCR and immunoblotting, we studied POMGNT1 expression at the mRNA and protein levels in various mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of its protein product in mouse retinal sections and in 661W cultured cells. The intranuclear distribution of POMT1 and POMT2, the two enzymes preceding POMGnT1 in the α-DG O-mannosyl glycosylation pathway, was also analyzed. RESULTS: POMGNT1 mRNA and its encoded protein were expressed in the neural retina of all mammals studied. POMGnT1 was located in the cytoplasmic fraction in the mouse retina and concentrated in the myoid portion of the photoreceptor inner segments, where the protein colocalized with GM130, a Golgi complex marker. The presence of POMGnT1 in the Golgi complex was also evident in 661W cells. However, and in contrast to retinal tissue, POMGnT1 additionally accumulated in the nucleus of the 661W photoreceptors. Colocalization was found within this organelle between POMGnT1 and POMT1/2, the latter associated with euchromatic regions of the nucleus. CONCLUSIONS: Our results indicate that POMGnT1 participates not only in the synthesis of O-mannosyl glycans added to α-DG in the Golgi complex but also in the glycosylation of other yet-to-be-identified proteins in the nucleus of mouse photoreceptors.

Human papillomavirus types in cases of squamous cell carcinoma of head and neck in Colombia.

UNLABELLED: Estimating the type-specific prevalence of human papillomavirus (HPV) in head and neck cancer (HNSCC) is helpful in predicting the impact of HPV immunization. OBJECTIVE: To estimate the overall prevalence, and gender and age-specific prevalence of HPV in HNSCC. METHOD: This cross sectional retrospective study was carried out in four pathology laboratories of Medellin, Colombia. HPV testing was performed by GP5+/6+ PCR-based RLB and HPV 16 and 18 type-specific PCR. RESULTS: 175 primary HNSCC cases consecutively diagnosed between 1999 and 2008 with confirmed diagnosis and amplifiable DNA were included. Overall HPV prevalence was 18.9%. HPV was found in 23.9%, 17.5% and 13.3% of the oral cavity, larynx and oropharynx cases respectively. Among HPV positive cases, 82% were HPV 16 and 18% were HPV 18. No other HPV genotypes were identified. Most patients were males. Male patients were younger that their female counterparts, particularly in oral cavity cancer cases. CONCLUSION: HPV 16 and 18 genotypes were found in nearly 20% of HNSCC cases in Colombian patients. The impact of HPV vaccination for the prevention of HNSCC in this population deserves further evaluation.

Genótipos de vírus de papiloma humano em carcinoma de células escamosas de cabeça e pescoço na Colômbia / Human papillomavirus types in cases of squamous cell carcinoma of head and neck in Colombia

Estimar a prevalência de tipos do vírus de papiloma humano (HPV) em câncer de cabeça e pescoço (CCP) é relevante para se prever o impacto da vacina contra o HPV. OBJETIVO: Estimar a prevalência global, por gênero e idade, do vírus do HPV em CCP. MÉTODO: Estudo transversal, retrospectivo envolvendo quatro laboratórios de patologia de Medellín, Colômbia. O exame utilizado foi o PCR GP5+/6+ e hibridização reversa. Além disso, os HPV 16 e 18 foram identificados utilizando-se PCR específica para esses tipos. RESULTADOS: Foram incluídos 175 casos primários de CCP, consecutivamente diagnosticados entre 1999 e 2008, com diagnóstico confirmado e DNA amplificado. A prevalência de HPV foi de 18,9%. O HPV foi encontrado em 23,9%, 17,5% e 13,3% dos casos de cavidade oral, laringe e orofaringe, respectivamente. Entre os casos de VPH+, 82% foram HPV 16 e 18% HPV18. A maioria dos casos foi de pessoas do sexo masculino. Nos homens, a idade de diagnóstico foi menor do que nas mulheres, principalmente naqueles de acometimento na cavidade oral. CONCLUSÃO: Os HPV 16 e 18 foram encontrados em quase 20% desses casos de CCP. O impacto da vacinação contra o HPV para a prevenção desse câncer na população merece maiores estudos.

Estimating the type-specific prevalence of human papillomavirus (HPV) in head and neck cancer (HNSCC) is helpful in predicting the impact of HPV immunization. OBJECTIVE: To estimate the overall prevalence, and gender and age-specific prevalence of HPV in HNSCC. METHOD: This cross sectional retrospective study was carried out in four pathology laboratories of Medellin, Colombia. HPV testing was performed by GP5+/6+ PCR-based RLB and HPV 16 and 18 type-specific PCR. RESULTS: 175 primary HNSCC cases consecutively diagnosed between 1999 and 2008 with confirmed diagnosis and amplifiable DNA were included. Overall HPV prevalence was 18.9%. HPV was found in 23.9%, 17.5% and 13.3% of the oral cavity, larynx and oropharynx cases respectively. Among HPV positive cases, 82% were HPV 16 and 18% were HPV 18. No other HPV genotypes were identified. Most patients were males. Male patients were younger that their female counterparts, particularly in oral cavity cancer cases. CONCLUSION: HPV 16 and 18 genotypes were found in nearly 20% of HNSCC cases in Colombian patients. The impact of HPV vaccination for the prevention of HNSCC in this population deserves further evaluation.

Distribución de variantes del virus del papiloma humano 16 (VPH 16) en mujeres con y sin neoplasia intraepitelial cervical grado 3 y cáncer cervical / Distribution of variants of the human papilloma virus 16 (HPV 16) in Women with and without grade 3 cervical intraepithelial neoplasia and cervical cancer

Objetivos: Describir la distribución de variantes del virus del papiloma humano 16 en mujeres con y sin neoplasia intraepitelial cervical grado 3 y cáncer cervical. Métodos: Se determinaron las variantes moleculares en casos de carcinoma escamocelular, adenocarcinoma cervical y en mujeres sin anormalidades citológicas de alto grado y positivas para el virus del papiloma humano 16. Para la detección de las variantes moleculares se amplificó el marco abierto de lectura del gen E6 del virus del papiloma humano 16 y se utilizó una técnica de hibridación reversa para la detección de los principales cambios de nucleótidos que identifican las ramas filogenéticas y las clases de variantes. Resultados:Hubo diferencias estadísticamente significativas en la distribución de variantes de virus del papiloma humano 16. Los controles no presentaron infecciones con variantes no europeas, mientras que ellas estuvieron presentes en el 30% de los casos de carcinoma escamocelular o neoplasia intraepitelial cervical grado tres. En adenocarcinoma, el 65% de las infecciones fueron del tipo no europeo. Conclusiones: La prevalencia de variantes no europeas de virus de papiloma humano 16 fue de 31,2% en neoplasia intraepitelial cervical grado 3 y cáncer escamocelular, y de 64,1% en adenocarcinoma de cérvix, mientras que estas no se observaron en mujeres sin cáncer.

Objectives: To describe the distribution of the variants of the human papilloma virus 16 in women with and without grade 3 cervical intraepithelial neoplasia and cervical cancer. Methods: Molecular variants were established in cases of squamous cell carcinoma, cervical adenocarcinoma and in women with high grade Pap smear abnormalities who tested positive for human papilloma virus 16. For the detection of molecular variants the open reading framework for the E6 gene of the human papilloma virus 16 was amplified and a reverse hybridization technique was utilized for the detection of major changes in the nucleotides which identify the phylogenetic branches and classes of variants. Results: There were statistically significant results in the distribution of the variants of the human papilloma virus 16. Control cases showed no infections with non European variants, but they were present in 30% of squamous cell carcinoma or grade three cervical intraepithelial neoplasia. For adenocarcinoma, 65% of infections were of non European type. Conclusions: The prevalence of non European variants of the human papilloma virus 16 was 31.2% in grade 3 cervical intraepithelial neoplasia and squamous cell cancer, and 64.1% in cervical adenocarcinoma; however, these were not observed among women without cancer.

Diagnosis of gestational, congenital, and placental malaria in Colombia: comparison of the efficacy of microscopy, nested polymerase chain reaction, and histopathology.

The technical capability of different methods to diagnose Plasmodium in maternal peripheral blood, placenta, and umbilical cord blood has not been assessed in Colombia and seldom explored in other malaria-endemic regions. We designed a study to compare the technical and the operational-economical performances of light microscopy (LM), nested polymerase chain reaction (nPCR), and histopathology (HP). In maternal blood, LM had 41% sensitivity and 100% specificity and in placental blood, 35% and 100%, respectively, compared with nPCR. In placental tissue, LM had 33% sensitivity and 95% specificity; and nPCR 47% and 77%, respectively; compared with HP. Light microscopy had the best operational-economical qualification. We concluded that nPCR and HP performed better compared with LM, but field implementation of these two techniques remains a problem. Therefore, LM is recommended as the gold standard for diagnosis of gestational malaria and placental blood infection in the field.