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ASPM is a mitotic protein expressed at low levels in normal tissues. We identified ASPM as being both mutated and amplified in early lesions and invasive breast cancer. 1We used qPCR and immunofluorescence to validate these results in cell lines, and then analyzed the effect of ASPM inhibition on growth of normal, premalignant and invasive breast cancer cells using growth assays and cell cycle analysis. Our results suggest that ASPM may be a biomarker for breast cancer forming potential, as well as a putative prophylactic target.
ASPM, abnormal spindle-like microcephaly-associated protein, is a mitotic spindle regulatory protein assisting in microtubule polymerization and centriole stabilization.2 It is largely expressed in embryonic tissues, and not in adult tissues.3,6 ASPM inactivation is associated with human neuropathies. ASPM upregulation has been observed in gliomas and in basal-like breast cancer. 6,7
We performed a screen to detect genes that were preferentially mutated in a subset of invasive breast cancer associated with poor patient survival1. We then performed a second screen to identify early drivers of aggressive breast cancer from the top thirty-two preferentially mutates genes. From this screen ASPM was the top candidate.
ASPM is upregulated in premalignant breast cells and is required for their growth.Conclusions
ASPM levels are low in normal breast cells but increase as stages of breast cancer progress.ASPM knockdown does not affect the growth or division of normal breast cells (hTERT).Knockdown of ASPM inhibits growth and affects cell division for both pre-malignant breast cancer cells (DCIS.com) and invasive breast cancer cells (MCF7), indicating ASPM is important for proliferation in these cell types.
ImplicationsIf ASPM is not required by normal breast cells compared to pre-malignant and invasive breast cells, then
ASPM may be a good biomarker for high risk lesionsASPM may be a target for breast cancer prevention
Materials/MethodsTransfections were performed using Dharmafect and Opti-Mem along with the appropriate siRNA treatment. RNA for qPCR was extracted using the Qiagen QiaShredder and RNeasy Mini Kits. cDNA was made using PT 200 Peltier Thermal Cycler. qRT-PCR was performed using 7900 HT Fast Real-Time PCR System and analyzed with Sequence Detection Systems (SDS) software.Cell cycle analysis was performed using the Accuri C6 Flow Cytometer. Data was analyzed using FlowJo software.Immunofluorescence used ASPM antibody (1:500) and goat-anti-rabbit antibody (488) as the primary and secondary antibodies, respectively. Slides were mounted using Vectashield Mounting Medium for Flouroescence with Dapi. Pictures were taken using a Nikon Eclipse Ti Microscope.
References1) Haricharan et al. BCRT. 2014.2) Xu et al. PlosOne. 2012.3) Fish et al. PNAS. 2006.4) Oncomine Database5) BioGPS Database6) Higgins et al. BMC Cell Biology. 2010.7) Zhong et al. Cell Cycle. 2005 ASPM Knockout Studies
The Role of ASPM in Normal, Pre-Malignant and Invasive Breast Cancer CellsKristen Maslar, Svasti Haricharan, Powel BrownCancer Prevention & Research Institute of Texas Summer Research ProgramDepartment of Clinical Cancer Prevention
1. Test whether ASPM is overexpressed in premalignant cells relative to normal breast cells using qRT-PCR and immunofluorescence.2. Test whether ASPM is required for the growth of pre-invasive, but not for the growth of normal breast cells using a growth assay.3. Test whether ASPM is required for mitosis in pre-invasive but not normal breast cells using cell cycle analysis.
ASPM Functional ProfileASPM gene expressionResultsFigure 4: Average ASPM mRNA levels within different types of cell lines were tested using qRT-PCR. ASPM levels are lowest in normal breast cells and increase as the disease progresses.ASPM Baseline LevelsFigure 3: Heat maps for five different studies display ASPM gene expression ranging from normal breast tissue to invasive breast cancer tissue. The blue indicates the lowest levels of ASPM, whereas the yellow indicates highest levels of ASPM4, 5.Recurrence-free Survival CurveFigure 2: This graph represents the probability of recurrence-free survival for breast cancer based on ASPM levels. Higher ASPM levels result in a worse recurrence-free survival opposed to lower survival with lower ASPM levels.Figure 1: The illustration represents the functional profile of ASPM. When a cell is not undergoing mitosis, ASPM remains in the cyptoplasm. When a cell is ready to undergo mitosis, however, ASPM can cycle into the nucleus to assist in various mitotic functions, resulting in proliferation and division.Figure 5: The set of graphs display both siASPM knockdown and its affect on cell growth. The graphs on the left indicate siASPM knockdown calculated from qRT-PCR for both hTERT (normal breast) and DCIS.com (pre-malignant) cells lines. The graphs on the right display a growth assay in the presence and absence of siASPM for both hTERT and DCIS.com cell lines.siLucsiASPMhTERTDCIS.comhTERTMCF7Figure 7: Cell cycle analysis was performed to show how the presence and absence of ASPM affects stages of the cell cycle. The top images represent graphs of normal breast cells with and without ASPM, indicating no difference between them. The bottom graphs represent normal breast cells (hTERT) and ER+ breast cancer cells (MCF7). hTERT cells show no difference in cell cycle regulation, whereas MCF7 show an increase in the G2/M phase, indicating ASPM is important for division in breast cancer cells.Figure 6: Immunofluorescence displays ASPM within normal breast cells (hTERT) and invasive breast cancer cells (MCF7). For the negative control (siLuc), ASPM is more pronounced in the MCF7 cells than in hTERT cells due to their different in baseline levels. For the positive control (siASPM), there is a reduction in ASPM in the MCF7 cells but no change in the hTERT cells.siLucsiASPMNo MitosisMitosis
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