EPMA position paper in cancer:current overview and future perspectives

At present, a radical shift in cancer treatment is occurring in terms of predictive, preventive, and personalized medicine (PPPM). Individual patients will participate in more aspects of their healthcare. During the development of PPPM, many rapid, specific, and sensitive new methods for earlier detection of cancer will result in more efficient management of the patient and hence a better quality of life. Coordination of the various activities among different healthcare professionals in primary, secondary, and tertiary care requires well-defined competencies, implementation of training and educational programs, sharing of data, and harmonized guidelines. In this position paper, the current knowledge to understand cancer predisposition and risk factors, the cellular biology of cancer, predictive markers and treatment outcome, the improvement in technologies in screening and diagnosis, and provision of better drug development solutions are discussed in the context of a better implementation of personalized medicine. Recognition of the major risk factors for cancer initiation is the key for preventive strategies (EPMA J. 4(1):6, 2013). Of interest, cancer predisposing syndromes in particular the monogenic subtypes that lead to cancer progression are well defined and one should focus on implementation strategies to identify individuals at risk to allow preventive measures and early screening/diagnosis. Implementation of such measures is disturbed by improper use of the data, with breach of data protection as one of the risks to be heavily controlled. Population screening requires in depth cost-benefit analysis to justify healthcare costs, and the parameters screened should provide information that allow an actionable and deliverable solution, for better healthcare provision

Complex Systems Analysis of Cell Cycling Models in Carcinogenesis

A new approach to the modular, complex systems analysis of nonlinear dynamics in cell cycling network transformations involved in carcinogenesis is proposed. Carcinogenesis is a complex process that involves dynamically inter-connected biomolecules in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments that form numerous inter-related pathways referred to as networks.
The variable biotopology of such dynamic networks is highly complex, and has a number of interesting properties that can be formally characterized at one level of organization by mathematical structures called 'biogroupoids'. 
One such family of pathways contains the cell cyclins. Cyclins are proteins that link several critical pro-apoptotic and other cell cycling/ division components, including the tumor suppressor gene TP53 and its product, the Thomsen-Friedenreich antigen (T antigen), Rb, mdm2, c-Myc, p21, p27, Bax, Bad and Bcl-2, which all play major roles in carcinogenesis of many cancers. A novel theoretical analysis is thus possible based on recently published studies of cyclin signaling, with special emphasis placed on the roles of cyclins D1 and E, suggests novel clinical trials and rational therapies of cancer through reestablishment of cell cycling inhibition in metastatic cancer cells

The role of ERBB3 inhibitors as cancers therapeutics

Cancer is the most fatal disease after cardiovascular disease with over 8.2 million deaths worldwide each year. Ever since the serendipitous discovery of mustard gas as an anti-cancer therapeutic in the 1940s, serious efforts have been put into discovering more chemotherapies. Chemotherapies can be categorized into different groups such as alkylating agents (cisplatin, cyclophosphamide), antimetabolites (5-fluorouracil, Ara-C) and mitotic inhibitors (taxanes, vinca alkoids) among others. While chemotherapies have proven to kill cancer cells by targeting cell division processes, over time, tumor cells can adapt and become resistant to these drugs. With a growing understanding of cell signaling networks, targeted therapies are being developed to overcome the issue of chemotherapy resistance. Targeted therapies are highly specific molecules that bind to a specific cellular protein or molecule and block signaling networks associated with biological processes. One of the most frequently dysregulated receptor systems in cancers is the receptor tyrosine kinase family with ErbB being one of the most studied receptors families. ErbB or HER receptors consists of four structurally related receptor tyrosine kinases namely, EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4. The ErbB family of receptors plays a major role in morphogenesis of the human body as well as various cellular responses such as cell growth, differentiation and proliferation. Overexpression and dysregulation of these receptors, particularly EGFR and HER2, have been linked to a number of cancers such as breast cancer, gastric cancer, ovarian cancer and non-small cell lung cancer, to name a few. One of the most successful therapies against ErbB related cancers have been targeted therapies. Targeted therapies for ErbB related cancers are of two kinds: (i) Small molecule tyrosine kinase inhibitors (such as erlotinib and gefitinib against EGFR) and, (ii) Monoclonal antibodies (such as trastuzumab against HER2 and cetuximab against EGFR). These drugs function either by inhibiting the kinase activity of the receptor and preventing phosophorylation of tyrosine residues, or binding to some other site on the extracellular domain of the receptor and preventing ligand binding and heterodimerization of ErbB monomers. These drugs have proven to have limited efficacy as monotherapy, but are more effective in combination with standard chemotherapies. However, tumor cells can adapt their signaling networks developing resistance to targeted therapies over the course of treatment and lead to cancer progression. While overexpression and dysfunction of EGFR and HER2 are implicated in most ErbB driven cancers, recent studies have found HER3 playing a pivotal role in inducing resistance to EGFR and HER2 targeted therapies in various cancers and has been found to be the most sensitive node in driving the PI3K pathway leading to tumorigenesis. Thus, there is an urgent need to develop drugs targeted against HER3 and bring them into the clinic. Since HER3 lacks kinase activity, only monoclonal antibodies can be developed against it. Currently, there are a number of molecules in clinical development that target HER3. For example, patritumab and MM-121 are humanized monoclonal antibodies that target the extracellular domain of HER3 receptor and leads to inhibition of HER3-PI3K signaling followed by rapid internalization of the receptor. MM-111 and MM-141, two different bispecific monoclonal antibodies that bind to HER2, HER3 and IGFR-1, HER3, respectively, are currently in clinical development. HER3 inhibitors provide hope to effectively overcome HER3 induced tumor resistance and successfully treat several ErbB driven cancers. However, further development of HER3 inhibitors is necessary by taking strategic approaches. One of these approaches it the utilization of systems biology, a branch of biology that involves computational and mathematical modeling of complex biological systems with the aim of discovering emergent properties of biological systems. Systems biology enables researchers to get a deeper understanding of biological networks such as that of ErbB and make predictive models and test outcomes. This approach was used by Merrimack Pharmaceuticals to develop novel monoclonal antibodies against HER3. Computational outcomes were successfully validated by in vitro and in vivo experiments. Thus, this suggests that systems biology might be the future of designing and developing HER3 inhibitors that would successfully overcome HER3 resistance and cancer progression

Complex Systems Analysis of Arrested Neural Cell Differentiation during Development and Analogous Cell Cycling Models in Carcinogenesis

A new approach to the modular, complex systems analysis of nonlinear dynamics of arrested neural cell Differentiation--induced cell proliferation during organismic development and the analogous cell cycling network transformations involved in carcinogenesis is proposed. Neural tissue arrested differentiation that induces cell proliferation during perturbed development and Carcinogenesis are complex processes that involve dynamically inter-connected biomolecules in the intercellular, membrane, cytosolic, nuclear and nucleolar compartments. Such 'dynamically inter-connected' biomolecules form numerous inter-related pathways referred to as 'molecular networks'. One such family of signaling pathways contains the cell cyclins. Cyclins are proteins that link several critical pro-apoptotic and other cell cycling/division components, including the tumor suppressor gene TP53 and its product, the Thomsen-Friedenreich antigen (T antigen), Rb, mdm2, c-Myc, p21, p27, Bax, Bad and Bcl-2, which play major roles in various neoplastic transformations of many tissues. The novel theoretical analysis presented here is based on recently published studies of arrested cell differentiation that normally leads to neural system formation during early developmental stages; the perturbed development may involve cyclin signaling and cell cycling responsible for rapidly induced cell proliferation without differentiation into neural cells in such experimental studies; special emphasis in this modular model is placed upon the roles of cyclins D1 and E, and does suggest novel clinical trials as well as rational therapies of cancer through re-establishment of cell cycling inhibition in metastatic cancer cells. Cyclins are proteins that are often over-expressed in cancerous cells (Dobashi et al., 2004). They may also be over-expressed in cells whose differentiation is arrested during the early stages of organismic development, leading to increased cell proliferation instead of differentiation into specialized tissues such as those forming the neural system. Cyclin-dependent kinases (CDK), their respective cyclins, and inhibitors of CDKs (CKIs) were identified as instrumental components of the cell cycle-regulating machinery. In mammalian cells the complexes of cyclins D1, D2, D3, A and E with CDKs are considered motors that drive cells to enter and pass through the “S” phase. Cell cycle regulation is a critical mechanism governing cell division and proliferation, and it is finely regulated by the interaction of cyclins with CDKs and CKIs, among other molecules (Morgan et al., 1995). A categorical and Topos framework for Łukasiewicz Algebraic Logic models of nonlinear dynamics in complex functional genomes and cell interactomes is also proposed. Łukasiewicz Algebraic Logic models of genetic networks and signaling pathways in cells are formulated in terms of nonlinear dynamic systems with n-state components that allow for the generalization of previous logical models of both genetic activities and neural networks. An algebraic formulation of varying 'next-state' functions is extended in a Łukasiewicz-Topos with an n-valued Łukasiewicz Algebraic Logic subobject classifier description that represents non-random and nonlinear network activities as well as their transformations in developmental processes and carcinogenesis. Important aspects of Cell Cycling, the Control of Cell Division,and the Neoplastic Transformation in Carcinogenesis are being considered and subjected to algebraic-logico- relational, and computer-aided investigations. The essential roles of various levels of c-Myc, p27 quasi-complete inhibition/blocking, TP53 and/or p53 inactivation, as well as the perpetual hTERT activation of Telomerase biosynthesis are pointed out as key conditions for Malignant Cell transformations and partial re-differentiation leading to various types of cancer such as lung, breast,skin, prostate and colon. Rational Clinical trials, Individualized Medicine and the potential for optimized Radio-, Chemo-, Gene-, and Immuno- therapies of Cancers are suggested on the basis of integrated complex systems biology modeling of oncogenesis, coupled with extensive genomic/proteomic and interactomic High-throughput/high-sensitivity measurements of identified, sorted cell lines that are being isolated from malignant tumors of patients undergoing clinical trials with adjuvant signaling drug therapies. The implications of the cyclin model for abnormal neural development during early development are being considered in this model that may lead to explanations of subsequent cognitive changes associated with abnormal neural cell differentiation in environmentally-affected embryos. This new model may also be relevant to detecting the onset of senescing neuron transformations in Alzheimer's and related diseases of the human brain in ageing populations at risk

AKT can modulate the in vitro response of HNSCC cells to irreversible EGFR inhibitors

Epidermal growth factor receptor (EGFR) is overexpressed in up to 90% of head and neck squamous cell carcinoma (HNSCC) tumors. Cetuximab is the first targeted (anti-EGFR) therapy approved for the treatment of HNSCC patients. However, its efficacy is limited due to primary and secondary resistance, and there is no predict biomarkers of response. New generation of EGFR inhibitors with pan HER targeting and irreversible action, such as afatinib and allitinib, represents a significant therapeutic promise. In this study, we intend to compare the potential cytotoxicity of two anti-EGFR inhibitors (afatinib and allitinib) with cetuximab and to identify potential predictive biomarkers of response in a panel of HNSCC cell lines. The mutational analysis in the eight HNSCC cell lines revealed an EGFR mutation (p.H773Y) and gene amplification in the HN13 cells. According to the growth inhibition score (GI), allitinib was the most cytotoxic drug, followed by afatinib and finally cetuximab. The higher AKT phosphorylation level was associated with resistance to anti-EGFR agents. Therefore, we further performed drug combinations with anti-AKT agent (MK2206) and AKT1 gene editing, which demonstrated afatinib and allitinib sensitivity restored. Additionally, in silico analysis of TCGA database showed that AKT1 overexpression was present in 14.7% (41/279) of HNSCC cases, and was associated with perineural invasion in advanced stage. In conclusion, allitinib presented a greater cytotoxic profile when compared to afatinib and cetuximab. AKT pathway constitutes a predictive marker of allitinib response and combination with AKT inhibitors could restore response and increase treatment success.FINEP (MCTI/FINEP/MS/SCTIE/DECIT-01/2013 - FPXII-BIOPLAT) and the Assistance Program and Incentive Research (PAIP), Barretos Cancer Hospital São Paulo, Brazil. The authors would like to acknowledge the technical support of Gabriela Lamberti in the clonogenic assays. A.L.C and R.M.R are recipients of a National Counsel of Technological and Scientific Development (CNPq) scholarship and O.C.M is recipient of a Portuguese Foundation for Science and Technology (FCT) scholarship (SFRH/BPD/108351/2015)info:eu-repo/semantics/publishedVersio

Role of PLCy1 in the resistance mechanism to Anti-EGFR therapy in metastatic colorectal cancer

Tese de Mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2017Tumor metastases are responsible for approximately 90% of all cancer-related deaths. Cetuximab (Cetx) is a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), which was recently approved for the treatment of metastatic colorectal cancer (mCRC). However, Cetx effectiveness is only about 20% due to the existence of multiple resistance mechanisms downstream of EGFR. KRAS mutations are recognized as a predictor of resistance to anti-EGFR treatment, nevertheless, 54% of wild-type KRAS patients still do not respond to this therapy. Therefore, there is a clear need for new biomarkers capable of accurately predict response to therapy. PLCγ1 is activated by direct binding and phosphorylation by EGFR and has been implicated in oncogenic signaling downstream of this receptor. PLCγ1 catalyzes the hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), involved in diverse cellular processes, such as cell proliferation, differentiation and motility. In this thesis, we investigate the contribution of PLCγ1 for the resistance mechanism to Cetuximab, in an in vitro and a clinical approach. Overall, our results show that PLCγ1 is highly expressed in Cetuximab-resistant colon cancer cell lines. PLCγ1 knockdown in resistant cell lines (CACO-2 and HT-29) was able to sensitize them to Cetx. Furthermore, PLCγ1 overexpression in the most sensitive cell line (SW48) confers increased Cetuximab resistance. Additionally, SW48 cell line that was continuously exposed to Cetx for five months shows a slightly increase in PLCγ1 expression when compared with parental control. Finally, immunohistochemical analysis of PLCγ1 in human CRC samples shows an association between increased PLCγ1 expression and poor progression-free survival of patients under Cetx treatment. Taking together, our results show a correlation between PLCγ1 expression levels and Cetuximab resistance, suggesting that PLCγ1 could be a predictive biomarker of EGFR resistance, helping selecting patients more likely to respond to this therapy.O cancro colo-rectal é o terceiro cancro mais incidente a nível mundial, com a quarta maior taxa de mortalidade. A elevada mortalidade associada a este tipo de cancro é essencialmente devida à acrescida dificuldade no tratamento da doença metastática. Nos últimos anos, o desenvolvimento e utilização de novos fármacos, como os anticorpos monoclonais dirigidos contra o recetor do fator de crescimento epidérmico (EGFR), têm aumentado a eficácia das terapêuticas convencionais em tumores metastáticos. No entanto, existe ainda um grande número de doentes que não responde a estas terapêuticas ou que acaba por desenvolver resistência às mesmas, após um período inicial de tratamento. Dado este cenário, torna-se cada vez mais urgente a procura de novos biomarcadores, mais sensíveis e específicos, que possam indicar com maior clareza quais os pacientes que beneficiam destas terapêuticas. O EGFR está implicado no desenvolvimento e progressão de múltiplos tumores, nomeadamente nos casos de cancro colo-rectal. A ativação deste recetor conduz à ativação de várias vias de sinalização celulares implicadas no controlo da sobrevivência celular, progressão do ciclo celular, angiogénese, migração e invasão/metastização. O Cetuximab (Cetx) é um anticorpo monoclonal direcionado especificamente contra o EGFR, que se liga à sua porção extracelular com uma afinidade superior à dos seus ligandos endógenos. Desta forma, o Cetx bloqueia a ligação dos ligandos ao EGFR, impedindo a ativação do recetor, o que se traduz na inibição das vias intracelulares e dos processos por elas regulados. Além disso, o Cetx induz a internalização do EGFR levando à diminuição dos recetores disponíveis na superfície celular. Finalmente, o Cetx permite ainda o reconhecimento das células tumorais pelas células efetoras imunitárias citotóxicas, desencadeando o processo de citotoxicidade mediada por células dependentes de anticorpo. Infelizmente, apenas um pequeno número de pacientes responde eficazmente a esta terapêutica. Mutações ativadoras no gene KRAS, que codifica para uma proteína a jusante na via de sinalização do EGFR, estão já identificadas como fortes indicadores de resistência ao Cetuximab, uma vez que ativam constitutivamente as vias intracelulares, de forma independente do recetor. Ainda assim, 54% dos doentes que não apresentam mutações neste gene desenvolvem resistência (intrínseca ou adquirida) a esta terapêutica. A ativação constitutiva de outros efetores intracelulares, tais como BRAF, PI3K e PLCγ, pode também constituir um mecanismo de resistência à terapia anti-EGFR, sendo que a PLCγ nunca foi anteriormente estudada neste contexto. Existem duas isoformas da PLCγ, a PLCγ1 e a PLCγ2, sendo que a primeira é amplamente expressa, estando presente em quase todos os tecidos, e a segunda é expressa essencialmente em células do sistema imune. Ambas as PLCγ são diretamente ativadas por recetores do tipo tirosina cinase, dos quais fazem parte o EGFR, e, quando ativas, são responsáveis pela conversão do fosfolípido de membrana fosfatidilinositol 4,5-bifosfato (PIP2) em dois mensageiros secundários, o diacilglicerol (DAG) e o inositol trifosfato (IP3). Estes mensageiros secundários são essenciais para a regulação de múltiplos processos celulares como proliferação, diferenciação, migração e angiogénese. Diversos estudos demonstraram que a PLCγ1 possui um papel importante no desenvolvimento e progressão tumoral, nomeadamente ao nível da migração celular. Neste sentido, o knockdown da expressão da PLCγ1 numa linha celular de carcinoma mamário (MDA-MB-231) inibiu o desenvolvimento de metástases pulmonares em modelo animal de ratinho. Por outro lado, a sobre-expressão de PLCγ1 foi observada em diversos tipos de tumores, incluindo colo-rectais, quando comparados com tecidos normais adjacentes, e foi associada a um pior prognostico e a um risco aumentado de desenvolvimento de metástases à distancia. Para além deste facto, mutações no gene PLCG1 foram recentemente associadas com o desenvolvimento de angiossarcomas e linfomas cutâneos de células T. Finalmente, apesar de pouco estudada no contexto de resistência à terapêutica, mutações no gene da PLCG2 (isoforma maioritariamente expressa em células hematopoiéticas) foram associadas ao mecanismo de resistência ao Ibrutinib, um inibidor da tirosina cinase de Bruton, no tratamento de leucemia linfocítica crónica. Tendo em conta o papel da PLCγ1 na regulação de processos celulares como a migração, invasão e progressão tumoral, e a sua estreita relação com o recetor EGFR, o principal objetivo deste trabalho é explorar a hipótese do envolvimento da PLCγ1 no mecanismo de resistência à terapêutica anti-EGFR. Para testar esta hipótese começou-se por avaliar a resposta de um painel de cinco linhas celulares de cancro colo-rectal (todas KRAS wild-type) ao tratamento com Cetuximab e correlacionar essa resposta com o nível de expressão da PLCγ1. Os nossos resultados mostram que os níveis de expressão da PLCγ1 estão aumentados nas linhas celulares mais resistentes ao Cetuximab, quando comparados com os níveis de expressão das linhas mais sensíveis. De seguida, foi realizada a sobre--expressão da PLCγ1 na linha mais sensível (SW48), enquanto que na linha mais resistente (CACO-2) foi realizado o knockdown da expressão da PLCγ1. O Knockdown da expressão da PLCγ1 na linha CACO-2 permitiu sensibilizá-la de forma significativa (p=0,0289) ao tratamento com Cetx. Por outro lado, a sobre-expressão da PLCγ1 na linha SW48 fez com que esta aumentasse a resistência ao tratamento com Cetuximab. A sobre-expressão de um mutante PLCγ1 constitutivamente ativo na sua função lipase (ΔSA), não mostrou diferenças na resposta ao Cetuximab quando comparada com o controlo. Estes resultados indicam um possível mecanismo de resistência independente da função lipase da PLCγ1. Simultaneamente, uma linha celular com sensibilidade intermedia ao Cetx (HT-29) mas com mutação ativadora do gene BRAF, foi também selecionada para se realizar o knockdown da expressão da PLCγ1. Mais uma vez, o knockdown da expressão da PLCγ1 permitiu também aumentar a sensibilidade das células ao Cetx (p=0,0222), mesmo na presença de uma mutação ativadora no gene do BRAF (V600E). Por fim, a linha SW48 foi também continuamente exposta a elevadas concentrações de Cetuximab por um período de cinco meses, a fim de avaliar um possível envolvimento da PLCγ1 na resistência adquirida ao Cetx. Neste caso, foi possível ver um aumento de expressão da PLCγ1, comparativamente com as células parentais não tratadas. De uma forma geral, os nossos resultados sugerem que o aumento da expressão desta proteína poderá estar associado não só a um mecanismo de resistência inato ao tratamento, mas também a um mecanismo adaptativo de resistência ao Cetx. Por fim, a expressão da PLCγ1 foi avaliada num grupo retrospetivo de amostras (n=25) de casos de carcinoma colo-rectal, provenientes do serviço de anatomia patológica do Hospital de Santa Maria. Estas amostras correspondem a amostras de tumores primários de doentes tratados com Cetuximab em contexto da doença metastática. A expressão da PLCγ1 foi avaliada por imunohistoquímica e os resultados foram analisados por um médico patologista, que realizou um score de intensidades de marcação. A elevada expressão da PLCγ1 foi significativamente associada (p=0,0460) a uma diminuição no tempo de sobrevivência livre de progressão, em doentes sob tratamento com Cetx. Observou-se também uma tendência entre maiores níveis de expressão de PLCγ1 e uma diminuição da sobrevivência global, sem, no entanto, existir significância estatística. Em conclusão, os resultados obtidos neste trabalho sugerem uma associação negativa entre os níveis de expressão da PLCγ1 e a resposta ao Cetuximab. Esta relação foi observada nos estudos in vitro e na avaliação de amostras de pacientes. O aumento da expressão da PLCγ1 pode também estar associado ao desenvolvimento de resistência adquirida ao Cetuximab. Demonstrar o envolvimento da PLCγ1 em mecanismos de resistência ao Cetuximab, e possivelmente a outras terapias anti-EGFR, poderá ter um grande impacto clínico no tratamento do cancro colo-rectal metastático, não só como potencial biomarcador preditivo de resposta à terapêutica, mas também como um possível novo alvo terapêutico

Distinguishing features of cetuximab and panitumumab in colorectal cancer and other solid tumors

Cetuximab and panitumumab are two distinct monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR), and both are widely used in combination with chemotherapy or as monotherapy to treat patients with RAS wild-type metastatic colorectal cancer. Although often considered interchangeable, the two antibodies have different molecular structures and can behave differently in clinically relevant ways. More specifically, as an immunoglobulin (Ig) G1 isotype mAb, cetuximab can elicit immune functions such as antibody-dependent cell-mediated cytotoxicity involving natural killer cells, T-cell recruitment to the tumor, and T-cell priming via dendritic cell maturation. Panitumumab, an IgG2 isotype mAb, does not possess these immune functions. Furthermore, the two antibodies have different binding sites on the EGFR, as evidenced by mutations on the extracellular domain that can confer resistance to one of the two therapeutics or to both. We consider a comparison of the properties of these two antibodies to represent a gap in the literature. We therefore compiled a detailed, evidence-based educational review of the known molecular, clinical, and functional differences between the two antibodies and concluded that they are distinct therapeutic agents that should be considered individually during treatment planning. Available data for one agent can only partly be extrapolated to the other. Looking to the future, the known immune activity of cetuximab may provide a rationale for this antibody as a combination partner with investigational chemotherapy plus immunotherapy regimens for colorectal cance