Article | Published:

Molecular Diagnostics

Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells

British Journal of Cancervolume 119pages11061117 (2018) | Download Citation

Subjects

Abstract

Background

Adhesion-mediated activation of FAK/ERK signalling pathway, enabled by the formation of filopodial protrusions (FLP), has been shown to be an important event for triggering of dormancy-to-proliferation switch and metastatic outgrowth of breast cancer cells (BCC). We studied the role of actin-binding protein profilin1 (Pfn1) in these processes.

Methods

Quantitative immunohistochemistry (IHC) of BC tissue microarray (TMA) and survival analyses of curated transcriptome datasets of BC patients were performed to examine Pfn1’s association with certain clinicopathological features. FLP formation and single cell outgrowth of BCC were assessed using a 3D matrigel culture that accurately predicts dormant vs metastatic outgrowth phenotypes of BCC in certain microenvironment. Gene expression studies were performed to identify potential biological pathways that are perturbed under Pfn1-depleted condition.

Results

Lower Pfn1 expression is correlated with lower nuclear grade of breast tumours and longer relapse-free survival of BC patients. Pfn1 depletion leads to defects in FLP and outgrowth of BCC but without impairing either FAK or ERK activation. Guided by transcriptome analyses, we further showed that Pfn1 depletion is associated with prominent SMAD3 upregulation. Although knockdown and overexpression experiments revealed that SMAD3 has an inhibitory effect on the outgrowth of breast cancer cells, SMAD3 knockdown alone was not sufficient to enhance the outgrowth potential of Pfn1-depleted BCC suggesting that other proliferation-regulatory pathways in conjunction with SMAD3 upregulation may underlie the outgrowth-deficient phenotype of BCC cells upon depletion of Pfn1.

Conclusion

Overall, these data suggest that Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC.

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Acknowledgements

This work was supported by grants from the National Institute of Health (R01 2CA108707) to P.R. and the National Medical Research Council and Ministry of Health to S.G. This project also used the University of Pittsburgh Chemical Biology Facility that is supported in part by award P30CA047904. We thank Dr. Dutch Boltz for image acquisition of the BC TMA.

Author information

Author notes

    • Chang Jiang

    Present address: Harvard Medical School, Boston, MA, USA

    • Zhijie Ding

    Present address: Janssen Scientific Affairs, New Jersey, Raritan, USA

    • Marion Joy

    Present address: NSABP, Pittsburgh, PA, USA

  1. These authors contributed equally: Souvik Chakraborty, Chang Jiang

Affiliations

  1. Bioengineering, University of Pittsburgh, Pittsburgh, USA

    • Souvik Chakraborty
    • , Chang Jiang
    • , David Gau
    • , Michael Oddo
    • , Zhijie Ding
    • , Marion Joy
    •  & Partha Roy
  2. Drug Discovery Institute, University of Pittsburgh, Pittsburgh, USA

    • Laura Vollmer
    •  & Andreas Vogt
  3. Case Western Reserve University, Cleveland, USA

    • William Schiemann
  4. Cell Biology, University of Pittsburgh, Pittsburgh, USA

    • Donna Beer Stolz
  5. Center for Computational Biology, Duke-NUS Medical School, Singapore, Singapore

    • Sujoy Ghosh
  6. Pathology, University of Pittsburgh, Pittsburgh, USA

    • Partha Roy

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Contributions

S.C., C.J., D.G., and M.O. designed and performed experiments, and wrote the manuscript. Z.D. and M.J. performed TMA analyses. L.V. and A.V. performed image acquisition and analyses. A.V. also wrote the manuscript. W.S. contributed to reagents and provided intellectual contribution. D.B.S. assisted with imaging. S.G. performed bioinformatics analyses and wrote the manuscript. P.R. was involved in the overall planning of the study and writing of the manuscript.

Competing interests

The authors declare no competing interests.

Availability of data and materials

Gene-specific survival outcomes in cancer patients were obtained through kmplot.com, a public domain data analysis tool that utilises curated genomic and clinical data available through Gene Expression Omnibus (GEO), European Genome-Phenome Archive (EGA) and The Cancer Gene Atlas (TCGA).

Ethics approval and consent to participate

No ethical approval and consent to participate were needed as the breast cancer TMA was purchased from Yale where the clinicopathological features of the cases were provided in a completely anonymous manner. The study was performed in accordance with the declaration of Helsinki.

Note

This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0)

Corresponding author

Correspondence to Partha Roy.

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DOI

https://doi.org/10.1038/s41416-018-0284-6