Identification of circadian-related gene expression profiles in entrained breast cancer cell lines

Abstract

Cancer cells have broken circadian clocks when compared to their normal tissue counterparts. Moreover, it has been shown in breast cancer that disruption of common circadian oscillations is associated with a more negative prognosis. Numerous studies, focused on canonical circadian genes in breast cancer cell lines, have suggested that there are no mRNA circadian-like oscillations. Nevertheless, cancer cell lines have not been extensively characterized and it is unknown to what extent the circadian oscillations are disrupted. We have chosen representative non-cancerous and cancerous breast cell lines (MCF-10A, MCF-7, ZR-75-30, MDA-MB-231 and HCC-1954) in order to determine the degree to which the circadian clock is damaged. We used serum shock to synchronize the circadian clocks in culture. Our aim was to initially observe the time course of gene expression using cDNA microarrays in the non-cancerous MCF-10A and the cancerous MCF-7 cells for screening and then to characterize specific genes in other cell lines. We used a cosine function to select highly correlated profiles. Some of the identified genes were validated by quantitative polymerase chain reaction (qPCR) and further evaluated in the other breast cancer cell lines. Interestingly, we observed that breast cancer and non-cancerous cultured cells are able to generate specific circadian expression profiles in response to the serum shock. The rhythmic genes, suggested via microarray and measured in each particular subtype, suggest that each breast cancer cell type responds differently to the circadian synchronization. Future results could identify circadian-like genes that are altered in breast cancer and non-cancerous cells, which can be used to propose novel treatments. Breast cell lines are potential models for in vitro studies of circadian clocks and clock-controlled pathways.

Dosing time dependent in vitro pharmacodynamics of Everolimus despite a defective circadian clock

“…However, cultured human mammary epithelial cells maintain an inner circadian clocks. 22,41,42 Using luciferase reporter technology, our results revealed also serum shock induced the rhythmical expression of Bmal1 promotor in non-tumorigenic epithelial cell line MCF-10A, supporting it was endowed with functional circadian clocks. But ER C MCF-7 cell did not display an evident circadian oscillations of core clock genes expression under the examination of luciferase reporter technology, qRT-PCR and western-blot.…”

“…[30][31][32][33][34] However, circadian rhythms are often altered in breast cancer patients [35][36][37] as well as in breast tumors and breast cancer cells lines. 22,[38][39][40][41][42] Alterations and desynchronization of molecular clock machinery on genetic and epigenetic level were observed in more aggressive breast cancer and those lacking estrogen receptor. Numerous studies have suggested that there are no mRNA circadian-like oscillations of canonical circadian genes in many breast cancer cell lines, either in ER C cells (such as MCF-7 or T47D) or in ERcells (such as HS578T and MDA-MB231).…”

“…46,47 Even in MCF-7 cells who did not possess one net clock gene rhythm, the serum shock could still induce rhythmic mRNA expressions of more than 400 genes. 41 Thus, in chronotherapy, how to take into account not only clock gene dependent rhythms but also clock gene independent rhythms will be a new potential clue for the future. Remarkably, the xenografting of MCF-7 cells into nude rats could re-establish a circadian pattern in Bmal1 transcription, suggesting that the importance of host circadian time cues in the circadian coordination of cancer cells in vivo.…”

Circadian Oscillations Persist in Low Malignancy Breast Cancer Cells

“…Oscillations of these and ancillary circadian genes have been found to be arrhythmic/absent in several disease models and tissues, including peripheral blood mononuclear cells from chronic myeloid leukemia, 2 colorectal liver metastases, 3 and prostate 4 and breast cancer cell lines. [5][6][7][8] In most of these cases, RT-qPCR, western blots, and immunohistochemistry were used to evaluate circadian changes. These assays result in less-detailed oscillation assessments because sampling frequency is typically 4-6 h and the experiment covers ~48 h; more time-points and collection over a longer duration are difficult to achieve.…”

“…Similar studies revealed that core clock gene transcripts are present, but do not oscillate in breast cancer cell lines. [6][7][8][9][10] As examples, in work using MCF7 and MDA-MB-231 (among other breast cancer) cells, RT-qPCR and DNA/miRNA microarray data show that core circadian genes BMAL1 and PER2 lack rhythmic oscillations, although some clock controlled genes (CCGs) and miRNAs are rhythmic. 6,8 Another study in MCF7 cells indicated that upon synchronization, some core clock genes…”