A control cell before and after compression to a height of 4 μm, without the addition of MG132.

Time stamps are in min:sec, scale bars are 5 μm, and video playback is 30 frames per second. See also Fig 3C. (MP4)</p

A cyclin D1-overexpressing cell before and after compression.

Time stamps are in min:sec, scale bars are 5 μm, and video playback is 30 frames per second. See also Fig 3A. (MP4)</p

Cyclin D1 overexpression protects against spindle pole fracture during compression.

(A) Confocal time-lapse images of control and cyclin D1-overexpressing cells undergoing compression, where the control spindle fractures around 60 min after compression onset (cartooned at right). The fractured poles of the control spindle are indicated in the final frame by white arrows. Tubulin is labeled with SiR-tubulin. Scale bars = 5 μm; time stamps are in minutes. (B) Spindles in cyclin D1-overexpressing cells fractured less often than control spindles during the 74 minutes of compression (**p = 0.0048, Fisher’s exact test). n = 57 control and 53 cyclin D1 spindles pooled from 13 and 12 independent days, respectively. (C) Confocal time-lapse images of a control spindle undergoing compression to a height of 4 μm, without the addition of MG132. After the spindle fractures between the 10 and 50 min time points, the cell enters anaphase and segregates chromosomes into 3 masses (cartooned below). Interpolar microtubule bundles connect the original pole to each of the fractured poles (white arrowheads), while no interpolar bundles connect the two poles resulting from the fracture (white arrow). The cytokinetic furrow is disrupted between the two fractured poles by the final 80 min timepoint (yellow arrow). Scale bars = 5 μm; time stamps are in minutes.</p

NuMA, but not HSET, Aurora A kinase, or TACC3, is upregulated in cyclin D1-overexpressing cells.

(A) Western blot of NuMA, TACC3, HSET, Aurora A kinase, and cyclin D1 levels in MCF10APuro (control) and MCF10ACyclin D1 cell lines. The overexpressed cyclin D1 is HA-tagged. Vinculin is shown as a loading control. (B-C) Quantification of NuMA, cyclin D1 (B), HSET, Aurora A kinase, and TACC3 levels (C) normalized to vinculin levels. Lines show mean ± standard deviation of three independent replicates.</p

Raw images of all western blots.

The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.</div

A control cell before and after compression.

Time stamps are in min:sec, scale bars are 5 μm, and video playback is 30 frames per second. See also Fig 3A. (MP4)</p

Source data for all graphs.

The mitotic spindle is the bipolar, microtubule-based structure that segregates chromosomes at each cell division. Aberrant spindles are frequently observed in cancer cells, but how oncogenic transformation affects spindle mechanics and function, particularly in the mechanical context of solid tumors, remains poorly understood. Here, we constitutively overexpress the oncogene cyclin D1 in human MCF10A cells to probe its effects on spindle architecture and response to compressive force. We find that cyclin D1 overexpression increases the incidence of spindles with extra poles, centrioles, and chromosomes. However, it also protects spindle poles from fracturing under compressive force, a deleterious outcome linked to multipolar cell divisions. Our findings suggest that cyclin D1 overexpression may adapt cells to increased compressive stress, possibly contributing to its prevalence in cancers such as breast cancer by allowing continued proliferation in mechanically challenging environments.</div

The cell compression assay is quantitatively reproducible.

(A) Schematic diagram of cell compression assay using a microfluidic device. Cells were compressed to a height of 5 μm using computer-controlled negative pressure over 4 min, and compression was sustained for 70 additional minutes. Cells were live-imaged throughout to monitor changes in spindle architecture. (B) Side (XZ) views of a control spindle, labeled with SiR-tubulin, before and after (at 74 min) compression. X and Z scale bars = 3 μm. (C) Between the control and cyclin D1 cell lines, spindle heights did not significantly differ before compression, and spindles were compressed to a similar final height (measured at 74 min). ns, not significant. (D) Spindle lengths before and 10 minutes after compression onset (ns, not significant). (E) Spindle widths before and 10 minutes after compression onset (*p = 0.028, **p = 0.00066). For C-E, two-sample t-tests were performed with n = 57 control and 53 cyclin D1 spindles (C) or n = 48 control and 52 cyclin D1 spindles (D and E), pooled from 13 and 12 independent days, respectively. Spindles were excluded from length and width analysis if both poles were not in focus in the same z-plane.</p

Cyclin D1 overexpression promotes aberrant spindle architectures.

(A) Western blot of α-tubulin and cyclin D1 levels in MCF10APuro (control) and MCF10ACyclin D1 cell lines. All images are from the same blot, with intervening lanes removed. (B) Representative confocal immunofluorescence images (maximum intensity projections) of spindles stained for α-tubulin (green), CREST (yellow), centrin (magenta), and Hoechst (blue), with spindle phenotypes cartooned (right). Magnifications of the centrioles at each spindle pole are shown at right. Scale bars = 3 μm. (C) Frequency of the three observed metaphase spindle phenotypes in each MCF10A cell line. The distribution of phenotypes differs between cyclin D1 and control cells (*p = 0.010, Fisher’s exact test), with the cyclin D1 line enriched in cells with supernumerary centrioles. (D) Number of kinetochores per spindle. Metaphase spindles in the cyclin D1 cell line had significantly more kinetochores (representing the number of chromatids) than the control line (****p = 2.32x10-14, Mann-Whitney U test). Lines indicate mean ± standard deviation. Inset shows a smaller range of kinetochore numbers. For C and D, n = 80 control spindles and 91 cyclin D1 spindles, each pooled from 3 independent experiments.</p

Protein Sialylation Regulates a Gene Expression Signature that Promotes Breast Cancer Cell Pathogenicity

Many mechanisms have been proposed for how heightened aerobic glycolytic metabolism fuels cancer pathogenicity, but there are still many unexplored pathways. Here, we have performed metabolomic profiling to map glucose incorporation into metabolic pathways upon transformation of mammary epithelial cells by 11 commonly mutated human oncogenes. We show that transformation of mammary epithelial cells by oncogenic stimuli commonly shunts glucose-derived carbons into synthesis of sialic acid, a hexosamine pathway metabolite that is converted to CMP-sialic acid by cytidine monophosphate <i>N</i>-acetylneuraminic acid synthase (CMAS) as a precursor to glycoprotein and glycolipid sialylation. We show that CMAS knockdown leads to elevations in intracellular sialic acid levels, a depletion of cellular sialylation, and alterations in the expression of many cancer-relevant genes to impair breast cancer pathogenicity. Our study reveals the heretofore unrecognized role of sialic acid metabolism and protein sialylation in regulating the expression of genes that maintain breast cancer pathogenicity