Stress is a necessary and functional part of human physiology. From responding to life-threatening situations to getting people out of bed in the morning, stress serves a major purpose in human survival. However, when consistent and high levels of stress are experienced, it can pose a threat to human health. One of the major mediators of physiological stress is a hormone called cortisol. Cortisol is a well-defined substance and its function in normal physiology is well understood. Scientific research indicates that consistent and high levels of this hormone may be an aid in cancer’s ability to evade the human immune response. Despite this, there is not much known about its relationship with cancer. I used immunofluorescence to determine cell-to-cell variability of vimentin expression and DNA content for cells that were exposed to cortisol at consistent and frequent doses overtime and those not exposed to cortisol to determine if cortisol altered the variability of vimentin expression and DNA content. I observed no change in the variability in vimentin expression across both cell conditions. I did observe variability in DNA content across both cell conditions, with more variability in the population affected by cortisol. These results suggest that there might be a relationship between the stress induced by cortisol, taking place at the genomic level but may have no impact on specific protein expression. Potential implications of the research conducted are looks to preventative medicine in the context of stress experienced by members of marginalized groups as a way of preventing cancer development.
Triple Negative Breast Cancer (TNBC), indicated by the absence of estrogen, progesterone and human epidermal growth factor receptor 2 (HER2), is the most aggressive form of breast cancer characterized by high rates of metastasis and low survival. Among those diagnosed with TNBC, 34% contain Inhibitor of Growth 4 (ING4) deletion that is associated with poor patient outcomes. We previously showed that ING4 negatively regulates NF-B in breast cancer. Previous studies show parthenolide, a compound found in feverfew (Tanacetum parthenium) to inhibit NF-B in cervical and gastric cancer. We hypothesized that parthenolide inhibits cytokine-induced activation of NF-B in ING4 deficient TNBC cells. To test the hypothesis, previously established vectors, v2, ING4 wildtype and v2h1, ING4-deleted were synthesized in MDA-MB 231, a TNBC cell line, using a CRISPR/Cas9 system. Inflammatory cytokines, IL-1 and TNF, were tested in ING4 wildtype or ING4 deleted cells for elicited phosphorylation of NF-B, proliferation, and migration in the presence or absence of parthenolide. The results showed that TNF or IL-1 induced translocation phosphorylation of NF-B regardless of ING4 deletion. ING4 inhibited proinflammatory cytokine induced pp65, consistent with previous studies demonstrating the negative regulation of NF-B in ING4-sufficent cell lines. We found the optimal working dose of parthenolide, 100nM, had no effect on cell proliferation in the presence or absence of IL-1. Parthenolide inhibited IL-1induced phosphorylation of NF-B regardless of ING4 deletion. Parthenolide inhibited TNF-induced phosphorylation of NF-B in ING4-deleted cell lines. Moreover, parthenolide induced migration of TNBC cells regardless of ING4 presence of absence. TNF and parthenolide treated samples in ING4-deleted cell lines were found to inhibit cell migration to basal level. These results demonstrate the difference in inhibitory mechanism of parthenolide in induced phosphorylation of NF-B through proinflammatory cytokines TNF or IL-1This is demonstrated by the exclusivity of parthenolide inhibition of TNF induced phosphorylation of NF-B in ING4-deleted TNBC cell line. In contrast, parthenolide inhibition of IL-1 induced phosphorylation of NF-B occurred regardless of ING4 deletion. These results may inhibit parthenolide as an alternative to those with ING4-deleted TNBC due to its role in inducing cancer phenotype cell migration.
Cancer is sometimes depicted as a reversion to single cell behavior in cells adapted to live in a multicellular assembly. If this is the case, one would expect that mutation in cancer disrupts functional mechanisms that suppress cell-level traits detrimental to multicellularity. Such mechanisms should have evolved with or after the emergence of multicellularity. This leads to two related, but distinct hypotheses: 1) Somatic mutations in cancer will occur in genes that are younger than the emergence of multicellularity (1000 million years [MY]); and 2) genes that are frequently mutated in cancer and whose mutations are functionally important for the emergence of the cancer phenotype evolved within the past 1000 million years, and thus would exhibit an age distribution that is skewed to younger genes. In order to investigate these hypotheses we estimated the evolutionary ages of all human genes and then studied the probability of mutation and their biological function in relation to their age and genomic location for both normal germline and cancer contexts.
We observed that under a model of uniform random mutation across the genome, controlled for gene size, genes less than 500 MY were more frequently mutated in both cases. Paradoxically, causal genes, defined in the COSMIC Cancer Gene Census, were depleted in this age group. When we used functional enrichment analysis to explain this unexpected result we discovered that COSMIC genes with recessive disease phenotypes were enriched for DNA repair and cell cycle control. The non-mutated genes in these pathways are orthologous to those underlying stress-induced mutation in bacteria, which results in the clustering of single nucleotide variations. COSMIC genes were less common in regions where the probability of observing mutational clusters is high, although they are approximately 2-fold more likely to harbor mutational clusters compared to other human genes. Our results suggest this ancient mutational response to stress that evolved among prokaryotes was co-opted to maintain diversity in the germline and immune system, while the original phenotype is restored in cancer. Reversion to a stress-induced mutational response is a hallmark of cancer that allows for effectively searching “protected” genome space where genes causally implicated in cancer are located and underlies the high adaptive potential and concomitant therapeutic resistance that is characteristic of cancer.
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.