A Kunitz-type inhibitor from tick salivary glands: a promising novel antitumor drug candidate

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Lonomia obliqua venom induces NF-κB activation and a pro-inflammatory profile in THP-1-derived macrophage

Envenomation caused by contact with Lonomia obliqua bristles is characterized by pain, an intense systemic proinflammatory reaction and disturbances in the coagulation cascade that can cause severe clinical manifestations and death. However, the role of immune system components in these effects is still poorly understood. In this study, we evaluated the cytotoxic effect of L. obliqua venom on THP-1-derived macrophages and its ability to modulate inflammatory markers, as well as the cytokine and chemokine release profile. Our results show that L. obliqua venom is able to directly exert a potent pro-inflammatory reaction in macrophages, characterized by the activation of the NF-κB transcription factor pathway, the expression of CD80 and CD83, and the release of pro-inflammatory mediators such as TNF-α, IL-1β, IL-6, IL-8 and CXCL10. These results suggest that macrophages can play an important role during the orchestration of the inflammatory response present in envenomation caused by Lonomia obliqua caterpillars.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model.

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

High CD90 (THY-1) expression positively correlates with cell transformation and worse prognosis in basal-like breast cancer tumors.

Breast cancer is the most prevalent cancer among women, with the basal-like triple negative (TNBC) being the most agressive one, displaying the poorest prognosis within the ductal carcinoma subtype. Due to the lack of adequate molecular targets, the diagnosis and treatment of patients with the TNBC phenotype has been a great challenge. In a previous work, we identified CD90/Thy-1 as being highly expressed in the aggressive high malignancy grade Hs578T basal-like breast tumor cell line, pointing to this molecule as a promising breast tumor marker, which should be further investigated. Here, CD90 expression was analyzed in human breast cancer samples and its functional role was investigated to better assess the oncogenic nature of CD90 in mammary cells. Quantification of CD90 expression in human breast cancer samples, by tissue microarray, showed that high CD90 positivity correlates with metastasis and poor patient survival in the basal-like subtype. The functional genetic approach, by overexpression in the CD90 cDNA in a basal-like normal mammary cell line (MCF10A) and knockdown in a highly malignant cell line (Hs578T), allowed us to demonstrate that CD90 is involved with several cellular processes that lead to malignant transformation, such as: morphological change, increased cell proliferation, invasiveness, metastasis and activation of the EGFR pathway. Therefore, our results reveal that CD90 is involved with malignant transformation in breast cancer cell lines and is correlated with metastasis and poor patient survival in the basal-like subtype, being considered as a promising new breast cancer target.

Presença de marcadores de células-tronco em linhagens celulares humanas de câncer de mama: possível valor em diagnóstico, prognóstico e terapía / Analysis of stem cells markers in human breast cancer cell lines

O câncer de mama é a doença maligna que mais acomete as mulheres no mundo. Apesar dos inúmeros tratamentos, o óbito se deve principalmente à doença metastática que pode se desenvolver a partir do tumor primário. Esta progressão tumoral decorre da dificuldade de se estabelecer um prognóstico mais preciso. Atualmente, a teoria de células iniciadoras de tumor vem sendo estudada para tentar explicar a biologia do câncer e descrever novos alvos para prognósticos e terapias. O carcinoma mamário foi o primeiro tumor sólido para o qual foi identificada uma subpopulação celular, definida como CD44+/CD24-, apresentando as características de células iniciadoras tumorais. Embora este fenótipo venha sendo muito utilizado para descrever as células iniciadoras tumorais de mama, muitos trabalhos tem questionado a relevância clínica desses marcadores, enfatizando que outros marcadores devem ser identificados. Assim, o objetivo deste trabalho é analisar e caracterizar marcadores de células-tronco que possam estar relacionados com o grau de malignidade no modelo de câncer de mama. Inicialmente, analisou-se a expressão de 10 marcadores de células-tronco em diferentes linhagens de câncer de mama que apresentam graus crescentes de malignidade. O CD90 foi selecionado devido à alta expressão desse marcador na linhagem mais agressiva Hs578T. Para a caracterização deste marcador, realizou-se ensaios funcionais, através do silenciamento do CD90 na linhagem tumorigênica Hs579T e sua superexpressão na linhagem não-tumorigênica MCF10A. As linhagens celulares geradas foram caracterizadas quanto ao crescimento celular, potencial invasivo e metastático. Foi possível observar que houve uma alteração da morfologia nas linhagens transformadas com o CD90 e, também, um maior tempo de dobramento na linhagem Hs578T-CD90- e um menor na MCF10A-CD90+. Além disso, a linhagem MCF10-CD90+ foi capaz de crescer independentemente de EGF. Através da análise da via EGF, foi possível observar que houve um aumento da expressão da forma fosforilada do receptor e dos fatores Erk, c-Jun, e Jnk na linhagem MCF10A-CD90+ e uma diminuição dos mesmos na linhagem Hs578T-CD90-. A análise da atividade do elemento responsivo do fator de transcrição AP1 comprovou que a via de EGF é funcional na linhagem MCF10-CD90+. Também foram analisados os marcadores de transição epitélio-mesenquimal, verificando-se aumento da expressão dos marcadores mesenquimais na linhagem MCF10A-CD90+ e diminuição na linhagem Hs578T-CD90-. Os ensaios in vitro de invasão mostraram que as células MCF10-CD90+ são capazes de migrar e invadir e as células Hs578T-CD90- apresentam diminuição significativa da habilidade de migração e invasão. Além disso, os ensaios de metástase in vitro e in vivo, mostraram que a superexpressão de CD90 levou à malignização das células MCF10A. Por outro lado, a linhagem Hs578T-CD90- apresentou menor potencial metastático in vitro. Portanto, neste trabalho, pela primeira vez, o CD 90 foi caracterizado funcionalmente como um marcador envolvido na transformação maligna do carcinoma mamário, contribuindo, assim, para melhor entendimento da biologia do câncer de mama e para que se possa desenvolver novas ferramentas de diagnóstico/prognóstico e novos protocolos clínicos e terapêuticos

Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols