Unlocking Overexpressed Membrane Proteins to Guide Breast Cancer Precision Medicine.

Breast cancer (BC) is a prevalent form of cancer affecting women worldwide. However, the effectiveness of current BC drugs is limited by issues such as systemic toxicity, drug resistance, and severe side effects. Consequently, there is an urgent need for new therapeutic targets and improved tumor tracking methods. This study aims to address these challenges by proposing a strategy for identifying membrane proteins in tumors that can be targeted for specific BC therapy and diagnosis. The strategy involves the analyses of gene expressions in breast tumor and non-tumor tissues and other healthy tissues by using comprehensive bioinformatics analysis from The Cancer Genome Atlas (TCGA), UALCAN, TNM Plot, and LinkedOmics. By employing this strategy, we identified four transcripts (LRRC15, EFNA3, TSPAN13, and CA12) that encoded membrane proteins with an increased expression in BC tissue compared to healthy tissue. These four transcripts also demonstrated high accuracy, specificity, and accuracy in identifying tumor samples, as confirmed by the ROC curve. Additionally, tissue microarray (TMA) analysis revealed increased expressions of the four proteins in tumor tissues across all molecular subtypes compared to the adjacent breast tissue. Moreover, the analysis of human interactome data demonstrated the important roles of these proteins in various cancer-related pathways. Taken together, these findings suggest that LRRC15, EFNA3, TSPAN13, and CA12 can serve as potential biomarkers for improving cancer diagnosis screening and as suitable targets for therapy with reduced side effects and enhanced efficacy.

Aptamer-Based Recognition of Breast Tumor Cells: A New Era for Breast Cancer Diagnosis.

Breast cancer is one of the leading causes of death among women worldwide and can be classified into four major distinct molecular subtypes based on the expression of specific receptors. Despite significant advances, the lack of biomarkers for detailed diagnosis and prognosis remains a major challenge in the field of oncology. This study aimed to identify short single-stranded oligonucleotides known as aptamers to improve breast cancer diagnosis. The Cell-SELEX technique was used to select aptamers specific to the MDA-MB-231 tumor cell line. After selection, five aptamers demonstrated specific recognition for tumor breast cell lines and no binding to non-tumor breast cells. Validation of aptamer specificity revealed recognition of primary and metastatic tumors of all subtypes. In particular, AptaB4 and AptaB5 showed greater recognition of primary tumors and metastatic tissue, respectively. Finally, a computational biology approach was used to identify potential aptamer targets, which indicated that CSKP could interact with AptaB4. These results suggest that aptamers are promising in breast cancer diagnosis and treatment due to their specificity and selectivity.

Fibromodulin Gene Variants (FMOD) as Potential Biomarkers for Prostate Cancer and Benign Prostatic Hyperplasia.

By the year 2050, the world's elderly population may increase exponentially, raising the rate of disease characteristic of this group, such as prostate cancer (PCa) and benign prostatic hyperplasia (BPH). Prostate disorders have a multifactorial etiology, especially age and genetic factors. Currently, PCa is the second most frequent neoplasm in the male population worldwide. The fibromodulin gene encodes a small leucine-rich proteoglycan (SLRP) which acts in the collagen fibrillogenesis pathway, cell adhesion, and modulation of TGF-ß signaling pathways, which has been recently associated with PCa. The present study sequenced the coding region of the FMOD in a sample of 44 PCa, 90 BPH, and 82 controls from a Brazilian population, and the results identified 6 variants: 2 missenses (p.(Tyr42Ser) and p.(Pro24Ala)); 3 synonymous (p.(His253=), p.(Asn353=), and p.(Glu79=)); and 1 intronic (c.980-114A>G). Of these, p.(Tyr42Ser), p.(Pro24Ala), and p.(Asn353=) are rare variants, and p.(Tyr42Ser) was predicted as potential pathogenic by the algorithms used here, in addition to not being observed in controls, suggesting that may be a potential biomarker for development of PCa and BPH. In conclusion, we identified for the first time, in Brazilian individuals with PCa and BPH, a potentially pathogenic variant in the analysis of FMOD gene. Further studies are needed to investigate the deleterious effect of this variant on the structure and/or function of the FMOD protein.

Biotechnological Evolution of siRNA Molecules: From Bench Tool to the Refined Drug.

The depth and versatility of siRNA technologies enable their use in disease targets that are undruggable by small molecules or that seek to achieve a refined turn-off of the genes for any therapeutic area. Major extracellular barriers are enzymatic degradation of siRNAs by serum endonucleases and RNAases, renal clearance of the siRNA delivery system, the impermeability of biological membranes for siRNA, activation of the immune system, plasma protein sequestration, and capillary endothelium crossing. To overcome the intrinsic difficulties of the use of siRNA molecules, therapeutic applications require nanometric delivery carriers aiming to protect double-strands and deliver molecules to target cells. This review discusses the history of siRNAs, siRNA design, and delivery strategies, with a focus on progress made regarding siRNA molecules in clinical trials and how siRNA has become a valuable asset for biopharmaceutical companies.

Precision Medicine: Technological Impact into Breast Cancer Diagnosis, Treatment and Decision Making.

Breast cancer is the most common cancer in women, impacting 2.1 million women each year. The number of publications on BC is much higher than for any other type of tumor, as well as the number of clinical trials. One of the consequences of all this information is reflected in the number of approved drugs. This review aims to discuss the impact of technological advances in the diagnosis, treatment and decision making of breast cancer and the prospects for the next 10 years. Currently, the literature has described personalized medicine, but what will the treatment be called for in the coming years?

Pieces of the Complex Puzzle of Cancer Cell Energy Metabolism: An Overview of Energy Metabolism and Alternatives for Targeted Cancer Therapy.

Over the past decades, several advances in cancer cell biology have led to relevant details about a phenomenon called the 'Warburg effect'. Currently, it has been accepted that the Warburg effect is not compatible with all cancer cells, and thus the process of aerobic glycolysis is now challenged by the knowledge of a large number of cells presenting mitochondrial function. The energy metabolism of cancer cells is focused on the bioenergetic and biosynthetic pathways in order to meet the requirements of rapid proliferation. Changes in the metabolism of carbohydrates, amino acids and lipids have already been reported for cancer cells and this might play an important role in cancer progression. To the best of our knowledge, these changes are mainly attributed to genetic reprogramming which leads to the transformation of a healthy into a cancerous cell. Indeed, several enzymes that are highly relevant for cellular energy are targets of oncogenes (e.g. PI3K, HIF1, and Myc) and tumor suppressor proteins (e.g. p53). As a consequence of extensive studies on cancer cell metabolism, some new therapeutic strategies have appeared that aim to interrupt the aberrant metabolism, in addition to influencing genetic reprogramming in cancer cells. In this review, we present an overview of cancer cell metabolism (carbohydrate, amino acid, and lipid), and also describe oncogenes and tumor suppressors that directly affect the metabolism. We also discuss some of the potential therapeutic candidates which have been designed to target and disrupt the main driving forces associated with cancer cell metabolism and proliferation.

Antitumor Activity of Lankacidin Group Antibiotics Is Due to Microtubule Stabilization via a Paclitaxel-like Mechanism.

Lankacidin group antibiotics show strong antimicrobial activity against various Gram-positive bacteria. In addition, they were shown to have considerable antitumor activity against certain cell line models. For decades, the antitumor activity of lankacidin was associated with the mechanism of its antimicrobial action, which is interference with peptide bond formation during protein synthesis. This, however, was never confirmed experimentally. Due to significant similarity to paclitaxel-like hits in a previous computational virtual screening study, we suggested that the cytotoxic effect of lankacidin is due to a paclitaxel-like action. In this study, we tested this hypothesis computationally and experimentally and confirmed that lankacidin is a microtubule stabilizer that enhances tubulin assembly and displaces taxoids from their binding site. This study serves as a starting point for optimization of lankacidin derivatives for better antitumor activities. It also highlights the power of computational predictions and their aid in guiding experiments and formulating rigorous hypotheses.

Chemical synthesis, pharmacological evaluation and in silico analysis of new 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives as potential anti-mitotic agents.

We have synthesized new, biologically active mono- and di-substituted 2,3,3a,4,5,6-hexahydrocyclopenta[c]pyrazole derivatives bearing electron withdrawing groups and electron donating groups. These derivative structures were characterized by their spectral and analytical data. The newly synthesized hexahydropyrazole analogues were evaluated for their in vitro anticancer activity against breast and lung cancer cell lines using a cytotoxicity bioassay. To understand their mechanism of action, tubulin binding assays were performed which pointed to their binding to microtubules in a mode similar to but not identical to colchicine, as evidenced by their KD value evaluation. Computational docking studies also suggested binding near the colchicine binding site on tubulin. These results were further confirmed by colchicine-binding assays on the most active compounds, which indicated that they bound to tubulin near but not at the colchicine site. The moderate cytotoxic effects of these compounds may be due to the presence of electron donating groups on the para-position of the phenyl ring, along with the hexahydropyrazole core nucleus. The observed anti-cancer activity based on inhibition of microtubule formation may be helpful in designing more potent compounds with a hexahydropyrazole moiety.

Toward precision medicine of breast cancer.

In this review, we report on breast cancer's molecular features and on how high throughput technologies are helping in understanding the dynamics of tumorigenesis and cancer progression with the aim of developing precision medicine methods. We first address the current state of the art in breast cancer therapies and challenges in order to progress towards its cure. Then, we show how the interaction of high-throughput technologies with in silico modeling has led to set up useful inferences for promising strategies of target-specific therapies with low secondary effect incidence for patients. Finally, we discuss the challenge of pharmacogenetics in the clinical practice of cancer therapy. All these issues are explored within the context of precision medicine.

Osteopontin-c mediates the upregulation of androgen responsive genes in LNCaP cells through PI3K/Akt and androgen receptor signaling.

Androgen receptor (AR) signaling is a key pathway modulating prostate cancer (PCa) progression. Several steps in this pathway have been investigated in order to propose novel treatment strategies for advanced PCa. Total osteopontin (OPN) has been described as a biomarker for PCa, in addition to its role in activating the progression of this tumor. Based on the known effects of the OPNc splice variant on PCa progression, the present study investigated whether this isoform can also modulate AR signaling. In order to test this, an in vitro model was used in which LNCaP cells were cultured in the presence of conditioned medium (CM) secreted by PCa cells overexpressing OPNc (OPNc-CM). The activation of AR signaling was evaluated by measuring the expression levels of AR-responsive genes (ARGs) using quantitative polymerase chain reaction and specific oligonucleotides. The data demonstrated that all nine tested ARGs (Fgf8, TMPRSS2, Greb1, Cdk2, Ndrg1, Cdk1, Pmepa1, Psa and Ar) are significantly upregulated in response to OPNc-CM compared with LNCaP cells cultured in CM secreted by control cells transfected with empty expression vector. The specific involvement of OPNc was demonstrated by depleting OPNc from OPNc-CM using an anti-OPNc neutralizing antibody. In addition, by using a phosphoinositide 3-kinase (PI3K)-specific inhibitor and AR antagonists, such as flutamide and bicalutamide, it was also observed that upregulation of ARGs in response to OPNc-CM involves PI3K signaling and depends on the AR. In conclusion, these data indicated that OPNc is able to activate AR signaling through the PI3K pathway and the AR. These data further corroborate our previous data, revealing the OPNc splice variant to be a key molecule that is able to modulate key signaling pathways involved in PCa progression.

Caracterização funcional das isoformas variantes de 'splicing' da osteopontina nos carcinomas de ovário e próstata / Functional characterization of osteopontin 'splicing' variant isoforms in ovarian and prostate carcinomas

"A osteopontina (OPN) é um dos transcritos que apresenta maiores aumentos no nível de expressão no câncer de ovário (CO) e próstata (CaP), e está envolvida na tumorigênese e metástase. O gene que codifica a OPN está sujeito à 'splicing' alternativo, gerando 3 mensagens, denominadas de OPNa, OPNb e OPNc. O objetivo deste projeto é a caracterização do perfil de expressão e do papel funcional das isoformas variantes de 'splicing' da OPN na tumorigênese e progressão tumoral no CO e CaP. A OPNc encontra-se especificamente expressa nos tumores malignos de ovário. No CaP, observamos que as isoformas apresentam maior nível de expressão na amostras de CaP quando comparado com o tumor benigno da próstata. A expressão diferencial das isoformas da OPN nas amostras tumorais e não tumorais de ovário e próstata identificam as isoformas como potenciais novos biomarcadores para estas neoplasias. Observamos que a OPNc, no modelo de ovário e próstata, e a OPNb, no modelo de próstata, apresentam efeitos estimulatórios sobre a proliferação, migração, invasão, formação de colônia e crescimento de tumores in vivo. A OPNc nos dois modelos tumorais estimula a proliferação celular da IOSE e RWPE-1, indicando que a OPNc apresenta características pró-tumorigênicas. Descrevemos também que os efeitos da OPNb e OPNc são mediados pela via de sinalização PI3K/Akt. Com base na observação de que tumores que superexpressam a OPNc apresentam altos níveis de expressão de marcadores típicos de angiogênese nos tumores ovarianos, tais como VEGF-A, Flk-1 e CD34, investigamos o papel funcional e o mecanismo molecular pelo qual a OPNc estimula a angiogênese no CO. Nossos dados demonstram que a OPNc secretada interage com receptores do tipo integrinas de forma RGD-dependente, possivelmente ativando a via de sinalização PI3K/Akt. Observamos também que a OPNc secretada e a via de sinalização PI3k/Akt modulam a expressão de VEGF-A, c-Fos e c-Jun e a fosforilação de c-Jun. Adicionalmente, observamos que o meio condicionado da OPNc induz a proliferação, migração e adesão das células endoteliais HUVEC, de forma a contribuir para a neovascularização. Através de PCR em tempo real, caracterizamos que a OPNc altera a expressão de 34 genes, no modelo de CO, e 16 genes, no CaP, essenciais para a transformação e progressão tumoral destas neoplasias. Os resultados gerados por este estudo contribuem para o melhor entendimento da biologia e dos mecanismos moleculares do CO e CaP. O papel crucial destas isoformas em distintas etapas da progressão destes tumores indicam a OPNb e OPNc como potenciais alvos terapêuticos para o CO e CaP"(AU)

"Osteopontin (OPN) is one of the proteins overexpressed in ovarian carcinoma (OC) and prostate cancer (PCa), and is involved in tumorigenesis and metastasis. Alternative splicing of OPN (OPN-SI) leads to 3 isoforms, OPNa, OPNb, and OPNc. This study aims to characterize the expression profile and functional roles of OPN isoforms in OC and PCa tumor progression. In OC, OPNc is specifically expressed in ovarian tumor samples. PCa tissue samples presented significantly higher levels of OPN-SI transcripts than in BPH specimens. OPN-SI mRNA expression were positively correlated with Gleason Score. The OPNc isoform was the most upregulated variant and the best marker to distinguish patients groups, presenting sensitivity and specificity of 90% and 100%, respectively. OPNc, in OC and PCa, and OPNb, in PCa, significantly activated OvCar-3 and PC-3 cell proliferation, migration, invasion, anchorage independent growth and tumor formation in vivo. Additionally, we have also shown that some of the OPNc-dependent protumorigenic roles are mediated by PI3K/Akt signaling pathway. OPNc stimulated immortalized ovarian and prostate epithelial cell proliferation, indicating a role for this isoform in OC and PCa tumorigenesis. Functional assays using OPNc conditioned medium and an anti-OPNc antibody have shown that most cellular effects observed here were promoted by the secreted OPNc. We identified that OPNc can affect the expression of genes involved in cancer pathways in these tumor models. Tumors formed by OvCar-3 OPNc-overexpressing cells present high expression levels of typical angiogenesis markers, as VEGF-A, VEGFR-2 and CD34. Based on this observation, we also investigated the molecular mechanisms by which OPNc stimulates angiogenic processes. Our data showed that OPNc overexpression activates VEGF-A expression and secretion, also through the PI3K/Akt pathway. This splicing isoform is also able to activate the expression of c-Fos, c-Jun and phospho-c-Jun. OPNc role on activating the phosphorylation of c-Jun is mediated by integrin receptor, in an RGD dependent manner. OPNc-conditioned medium is able to induce HUVEC endothelial cells proliferation, migration and adhesion. According to our data, contributes to the physiopathology of ovarian and prostate cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches that selectively down regulate OPNc, altering its properties favoring OC and PCa tumor progression"(AU)