Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will ...provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.
Abstract The behaviour of cells cultured within three-dimensional (3D) structures rather than onto two-dimensional (2D) culture plastic more closely reflects their in vivo responses. Consequently, 3D ...culture systems are becoming crucial scientific tools in cancer cell research. We used a novel 3D culture concept to assess cell–matrix interactions implicated in carcinogenesis: a synthetic hydrogel matrix equipped with key biomimetic features, namely incorporated cell integrin-binding motifs ( e.g. RGD peptides) and the ability of being degraded by cell-secreted proteases ( e.g. matrix metalloproteases). As a cell model, we chose epithelial ovarian cancer, an aggressive disease typically diagnosed at an advanced stage when chemoresistance occurs. Both cell lines used (OV-MZ-6, SKOV-3) proliferated similarly in 2D, but not in 3D. Spheroid formation was observed exclusively in 3D when cells were embedded within hydrogels. By exploiting the design flexibility of the hydrogel characteristics, we showed that proliferation in 3D was dependent on cell-integrin engagement and the ability of cells to proteolytically remodel their extracellular microenvironment. Higher survival rates after exposure to the anti-cancer drug paclitaxel were observed in cell spheroids grown in hydrogels (40–60%) compared to cell monolayers in 2D (20%). Thus, 2D evaluation of chemosensitivity may not reflect pathophysiological events seen in patients. Because of the design flexibility of their characteristics and their stability in long-term cultures (28 days), these biomimetic hydrogels represent alternative culture systems for the increasing demand in cancer research for more versatile, physiologically relevant and reproducible 3D matrices.
The 15 members of the kallikrein-related serine peptidase (KLK) family have diverse tissue-specific expression profiles and putative proteolytic functions. The kallikrein family is also emerging as a ...rich source of disease biomarkers with KLK3, commonly known as prostate-specific antigen, being the current serum biomarker for prostate cancer. The kallikrein locus is also notable because it is extraordinarily responsive to steroids and other hormones. Indeed, at least 14 functional hormone response elements have been identified in the kallikrein locus. A more comprehensive understanding of the transcriptional regulation of kallikreins may help the field make more informed hypotheses about the physiological functions of kallikreins and their effectiveness as biomarkers. In this review, we describe the organization of the kallikrein locus and the structure of kallikrein genes and proteins. We also focus on the transcriptional regulation of kallikreins by androgens, progestins, glucocorticoids, mineralocorticoids, estrogens, and other hormones in animal models and human prostate, breast, and reproductive tract tissues. The interaction of the androgen receptor with androgen response elements in the promoter and enhancer of KLK2 and KLK3 is also summarized in detail. There is evidence that all kallikreins are regulated by multiple nuclear receptors. Yet, apart from KLK2 and KLK3, it is not clear whether all kallikreins are direct transcriptional targets. Therefore, we argue that gaining more detailed information about the mechanisms that regulate kallikrein expression should be a priority of future studies and that the kallikrein locus will continue to be an important model in the era of genome-wide analyses.
Kallikrein-related serine peptidases have diverse tissue- and disease-specific expression profiles and proteolytic functions. A prominent member of the kallikrein family, prostate-specific antigen, is the current serum biomarker for prostate cancer and is commonly used as a model target gene to study the actions of the androgen receptor. This review focuses on the regulation of kallikrein gene expression by steroid hormone receptors and other nuclear receptors as well as the structure, evolution and proteolytic functions of the kallikrein family.
Metabolic reprogramming is a hallmark of cancer. MicroRNAs (miRNAs) have been found to regulate cancer metabolism by regulating genes involved in metabolic pathways. Understanding this layer of ...complexity could lead to the development of novel therapeutic approaches.
miRNAs are noncoding RNAs that have been implicated as master regulators of gene expression. Studies have revealed the role of miRNAs in the metabolic reprogramming of tumor cells, with several miRNAs both positively and negatively regulating multiple metabolic genes. The tricarboxylic acid (TCA) cycle, aerobic glycolysis, de novo fatty acid synthesis, and altered autophagy allow tumor cells to survive under adverse conditions. In addition, major signaling molecules, hypoxia-inducible factor, phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin/phosphatase and tensin homolog, and insulin signaling pathways facilitate metabolic adaptation in tumor cells and are all regulated by miRNAs. Accumulating evidence suggests that miRNA mimics or inhibitors could be used to modulate the activity of miRNAs that drive tumor progression via altering their metabolism. Currently, several clinical trials investigating the role of miRNA-based therapy for cancer have been launched that may lead to novel therapeutic interventions in the future.
In this review, we summarize cancer-related metabolic pathways, including glycolysis, TCA cycle, pentose phosphate pathway, fatty acid metabolism, amino acid metabolism, and other metabolism-related oncogenic signaling pathways, and their regulation by miRNAs that are known to lead to tumorigenesis. Further, we discuss the current state of miRNA therapeutics in the clinic and their future potential.
Single nucleotide polymorphisms (SNPs) have been classically used for dissecting various human complex disorders using candidate gene studies. During the last decade, large scale SNP analysis, i.e. ...genome-wide association studies (GWAS) have provided an agnostic approach to identify possible genetic loci associated with heterogeneous disease such as cancer susceptibility, prognosis of survival or drug response. Further, the advent of new technologies, including microarray-based genotyping as well as high throughput next generation sequencing has opened new avenues for SNPs to be used in clinical practice. It is speculated that the utility of SNPs to understand the mechanisms, biology of variable drug response and ultimately treatment individualization based on the individual's genome composition will be indispensable in the near future. In the current review, we discuss the advantages and disadvantages of the clinical utility of genetic variants in disease risk-prediction, prognosis, clinical outcome and pharmacogenomics. The lessons and challenges for the utility of SNP-based biomarkers are also discussed, including the need for additional functional validation studies.
Biophysical and biochemical properties of the microenvironment regulate cellular responses such as growth, differentiation, morphogenesis and migration in normal and cancer cells. Since ...two-dimensional (2D) cultures lack the essential characteristics of the native cellular microenvironment, three-dimensional (3D) cultures have been developed to better mimic the natural extracellular matrix. To date, 3D culture systems have relied mostly on collagen and Matrigel™ hydrogels, allowing only limited control over matrix stiffness, proteolytic degradability, and ligand density. In contrast, bioengineered hydrogels allow us to independently tune and systematically investigate the influence of these parameters on cell growth and differentiation. In this study, polyethylene glycol (PEG) hydrogels, functionalized with the Arginine-glycine-aspartic acid (RGD) motifs, common cell-binding motifs in extracellular matrix proteins, and matrix metalloproteinase (MMP) cleavage sites, were characterized regarding their stiffness, diffusive properties, and ability to support growth of androgen-dependent LNCaP prostate cancer cells. We found that the mechanical properties modulated the growth kinetics of LNCaP cells in the PEG hydrogel. At culture periods of 28 days, LNCaP cells underwent morphogenic changes, forming tumor-like structures in 3D culture, with hypoxic and apoptotic cores. We further compared protein and gene expression levels between 3D and 2D cultures upon stimulation with the synthetic androgen R1881. Interestingly, the kinetics of R1881 stimulated androgen receptor (AR) nuclear translocation differed between 2D and 3D cultures when observed by immunofluorescent staining. Furthermore, microarray studies revealed that changes in expression levels of androgen responsive genes upon R1881 treatment differed greatly between 2D and 3D cultures. Taken together, culturing LNCaP cells in the tunable PEG hydrogels reveals differences in the cellular responses to androgen stimulation between the 2D and 3D environments. Therefore, we suggest that the presented 3D culture system represents a powerful tool for high throughput prostate cancer drug testing that recapitulates tumor microenvironment.
Advances in tissue engineering have traditionally led to the design of scaffold- or matrix-based culture systems that better reflect the biological, physical and biochemical environment of the ...natural extracellular matrix. Although their clinical applications in regenerative medicine tend to receive most of the attention, it is obvious that other areas of biomedical research could be well served by the powerful tools that have already been developed in tissue engineering. In this article, we review the recent literature to demonstrate how tissue engineering platforms can enhance in vitro and in vivo models of tumorigenesis and thus hold great promise to contribute to future cancer research.
Emerging evidence suggests that gamma-tocotrienol (γ-T3), a vitamin E isomer, has potent anti-cancer properties against a wide-range of cancers. γ-T3 not only inhibited the growth and survival of ...cancer cells in vitro, but also suppressed angiogenesis and tumour metastasis under in vivo conditions. Recently, γ-T3 was found to target cancer stem cells (CSCs), leading to suppression of tumour formation and chemosensitisation. Despite its promising anti-cancer potential, the exact mechanisms responsible for the effects of γ-T3 are still largely unknown. Here, we report the identification of Ang-1 (Angiopoietin-1)/Tie-2 as a novel γ-T3 downstream target. In prostate cancer cells, γ-T3 treatment leads to the suppression of Ang-1 at both the mRNA transcript and protein levels. Supplementing the cells with Ang-1 was found to protect them against the anti-CSC effect of γ-T3. Intriguingly, inactivation of Tie-2, a member receptor that mediates the effect of Ang-1, was found to significantly enhance the cytotoxic effect of γ-T3 through activation of AMP-activated protein kinase (AMPK) and subsequent interruption of autophagy. Our results highlighted the therapeutic potential of using γ-T3 in combination with a Tie-2 inhibitor to treat advanced prostate cancer.
Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Theranostics, a combination of diagnostics and therapeutics, is an emerging concept in the field of precision ...medicine, and microRNAs (miRNAs) are predictive pioneers in this area.
miRNAs are small endogenous noncoding RNA molecules that regulate gene expression posttranscriptionally by targeting messenger RNAs. More than 60% of all protein coding genes are controlled by miRNAs, which makes them powerful regulators of the different cellular processes involved in the pathogenesis of various types of cancer, including prostate cancer. Growing evidence indicates the differential expression of miRNAs in tumor tissues. In addition, miRNAs in body fluids, known as circulating miRNAs, are present in remarkably stable forms and their alteration in prostate cancer has been well documented. Circulating miRNAs are known to originate from tumor tissues, thereby enabling intercellular communication via carriers to promote tumorigenesis and malignancy. In addition, fueled by recent advances, the use of miRNA-based anticancer therapies has been proposed with the onset of early phase clinical trials to assess the therapeutic efficacy of miRNAs.
In this review, we summarize the theranostic utility of miRNAs and outline their diagnostic and prognostic potential in prostate cancer. In addition, we discuss the current detection methodologies and emerging innovative strategies for the detection of miRNAs in body fluids and tumor tissues in the clinical setting. We also provide insight into the current and future therapeutic potential of miRNAs in prostate cancer.
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