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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster I5 - The Effect of HSPB1 Silencing on Cell Migration Dynamics

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Breast cancer is the most common cancer in the U.S., highlighting the need for deeper insights into the mechanisms that drive metastasis. For cancer cells to metastasize they must navigate through the basement membrane and enter the bloodstream. During this process, their nuclear lamina – composed of carefully folded Lamin A bundles – becomes deformed due to physical stress, disrupting gene expression and cell signaling.

Our research focuses on HSPB1, a stress-response protein chaperone that maintains the folded integrity of the nuclear lamina. Recent data from our lab shows that overexpression of Lamin A in breast cancer cells induces HSPB1 expression (Bell et al., 2022). Both Lamin A and HSPB1 levels are often altered in breast cancer, suggesting a potential interplay of these proteins in breast cancer progression.

Epithelial-mesenchymal transition (EMT) is a key player in cancer metastasis. The EMT process is activated at the invasive edge of primary tumors, allowing cancer cells to acquire invasive properties and spread to distant organs. EMT bears striking similarities to the wound-healing process: both require cellular migration, loss of epithelial traits, ECM remodeling, and share common signaling pathways. These molecular and morphological changes are driven by EMT transcriptional regulators Slug, Sox-9, Snail, Zeb, and Twist.

We examine whether HSPB1 and Lamin A are linked to EMT, aiming to elucidate how stress-related changes in the nuclear lamina affect cancer behavior and metastasis. Ultimately, unraveling a connection between HSPB1 and Lamin A in EMT could uncover new therapeutic targets to improve breast cancer treatment outcomes.

Materials and

Methods:  We engineered BT-549 breast cancer cells to overexpress Lamin A (BT-549-LMNA), along with mock-controls. To test whether HSPB1 was involved in BT-549 LMNA nuclear lamina mechanics, we utilized a cell-compression assay to measure changes in nuclear HSPB1 levels following mechanical stress. Cells were seeded into 6-well plates at 100,000 cells/cm² and compressed to 5 µm for 30 minutes with beads.

We transfected siHSPB1 into BT-549-LMNA cells using lipofection. siHSPB1 and lipofectamine concentrations were optimized to maximize silencing efficiency while minimizing toxicity. Western blotting confirmed HSPB1 silencing and Lamin A overexpression.

Following silencing, migration assays were performed to evaluate the cells’ metastatic potential. Wounds were created in a confluent monolayer using a pipette tip. Cells were incubated in RPMI with 1% FBS to minimize proliferation. Images were captured at specified timepoints over 24 hours. To quantify wound closure, the average wound width was measured at 3 locations per timepoint using ImageJ software.

To optimize EMT induction, MCF-7 cells were treated at different concentrations of TGFB. Myofibroblast markers Col I and III were used to validate the transformation. To track EMT, a flow cytometry panel with EMT markers CD24, CD44, CD104, and EpCAM is used. This panel has been validated with MCF-7 cells, which undergo EMT in the presence of TGFB. This induction method and flow cytometry will be employed in the BT-549 cells next. The flow cytometry panel will be used to monitor changes in EMT rates upon HSPB1 silencing in BT-549-LMNA cells, elucidating HSPB1’s involvement in EMT.

Results, Conclusions, and Discussions: To establish a relationship between HSPB1 and Lamin A, we cultured BT-549 breast cancer cells overexpressing Lamin A (BT-549-LMNA) (Fig. 1A, B). To confirm HSPB1’s function as a nuclear chaperone, we compressed BT-549-LMNA and mock-control cells and observed a 1.5-fold increase in nuclear HSPB1 levels (Fig. 1C). This suggested that HSPB1 translocates to the nucleus upon mechanical stress, possibly to organize the nuclear lamina.

We performed wound-healing assays with BT-549-LMNA cells to measure their migration. BT-549-LMNA cells exhibited faster wound closure compared to BT-549 cells, suggesting that Lamin A overexpression enhances breast cancer cell migration in wound-healing assays (Fig. 2A).
To study the role of HSPB1 in migration, we silenced HSPB1 using RNAi in BT-549-LMNA (Fig. 1D, E). Silencing HSPB1 in BT-549-LMNA cells slowed wound-healing compared to BT-549-LMNA cells (Fig. 2B). This indicates that HSPB1 could aid in facilitating EMT, as its depletion impaired migration. However, expression analysis of specific EMT markers must be performed to confirm this.

For further evidence linking HSPB1 and Lamin A, we tested how HSPB1 silencing affected Lamin A levels. Silencing HSPB1 in BT-549-LMNA cells led to a transient decrease in Lamin A levels at 48 hours but resulted in an increase in Lamin A levels by 72 hours (Fig. 1F). The initial drop in Lamin A could explain the slower migration observed in siHSPB1–BT-549-LMNA cells (Fig. 2B). We hypothesize that the rise in Lamin A levels by 72 hours could enhance cell migration.

We expect short-term and long-term HSPB1 silencing to impact cell migration differently. In our future work, we will allow the siHSPB1–BT-549-LMNA cells to proliferate for 72 hours prior to wound-healing experiments. Additionally, we will assess HSPB1 and Lamin A levels at different EMT stages using flow cytometry. To validate our EMT induction method, we conducted TGFB treatment at 5 ng/ml in MCF-7 cells, which are commonly used in EMT studies, and successfully induced EMT marker expression (Fig. 1G).

Overall, our study highlights HSPB1’s potential role in regulating EMT through interactions with Lamin A. Uncovering this mechanism could lead to novel therapeutic strategies, offering new avenues for breast cancer treatment.

Acknowledgements (Optional):  I would like to express my deepest appreciation to both my mentors, Alice and Sarah, who worked alongside me and became amazing guides on my journey to become a critical, independent thinker. Additionally, I am deeply indebted to Jan, who is a true cognoscente of the mechanobiology field, and has continuously supported me en route to becoming a young scientist. Finally, thank you to all Lammerding lab members who made this project and my undergraduate years in the lab a meaningful and memorable experience.