Contribution of HMHA1/ARHGAP45 Expression Induced by Hypoxia and Post-Radiation Reoxygenation to Cancer Cell Invasion: An HIF-Dependent Mechanism

Background and Motivation

Prior studies have extensively demonstrated that cancer cells in solid tumors acquire malignant characteristics such as invasiveness and treatment resistance under hypoxic conditions, leading to poor patient prognosis (References [3], [6]). Hypoxia-Inducible Factor (HIF) plays a central role in regulating metabolic enzymes and is involved in the control of many key factors responding to hypoxia.

Specifically, after radiation therapy, cancer cells surviving in severely hypoxic regions undergo post-radiation reoxygenation, leading to enhanced invasiveness through HIF-dependent mechanisms and further migration to near tumor blood vessels, resulting in recurrence. However, the specific role of HIF in mediating the invasiveness changes in cancer cells post-reoxygenation remains unclear. This study addresses this scientific question by investigating in-depth how HIF-dependent mechanisms influence the invasiveness of cancer cells in severely hypoxic and post-radiation reoxygenated environments.

Sources of Study

This study was conducted by Peter W. T. Lee, Tatsuya Suwa, Minoru Kobayashi, Hui Yang, Lina R. Koseki, Satoshi Takeuchi, Christalle C. T. Chow, Takaaki Yasuhara, and Hiroshi Harada, and published in the 2024 issue of the British Journal of Cancer. Authors are affiliated with the Cancer Cell Biology Laboratory at the Graduate School of Life Sciences, Kyoto University, the Division of Gene Repair Kinetics, the Center for Radiation Biology, the Department of Oncology at the University of Oxford, the Gene Stress Response Laboratory at Kyoto University, and the Division of Advanced Effects Research.

Methodology

Research Design and Process Overview

Researchers employed various molecular and cell biology techniques to identify the mechanism of HMHA1 expression induced under hypoxia and its functional roles. The research process is as follows:

Step One: Identifying HMHA1 Expression Induction Mechanism

Using gene chip and RNA-seq analysis, researchers compared gene expression profiles of cancer cells under normoxic and severely hypoxic conditions, screening for potential invasion-related genes specifically upregulated under hypoxia. RT-qPCR and Western Blot analyses revealed significant increases in HMHA1 mRNA and protein levels under specific oxygen concentrations (e.g., 3%, 1%, and less than 0.1% oxygen). Further, using HeLa tumor xenograft models in mice, they confirmed HMHA1 expression in vivo under hypoxic conditions through drug treatments.

Step Two: Regulation of HMHA1 Transcription Initiation and HIF Pathway Dependency

To verify if the transcription initiation of HMHA1 is induced by hypoxia, researchers used RNA polymerase inhibitor Actinomycin D, combined with HIF inhibitors (e.g., DFO and DMOG) knockdown and rescue experiments. These experiments demonstrated that HMHA1 expression is predominantly governed by the HIF pathway, with a compensatory mechanism between HIF-1 and HIF-2 identified.

Step Three: Role of HMHA1 in Cancer Cell Invasion

Using Boyden Chamber invasion assays, it was found that overexpression of HMHA1 enhanced cancer cell invasiveness under normoxic conditions. Under hypoxic conditions, inhibiting HMHA1 expression significantly reduced cancer cell invasiveness. Additionally, gelatin degradation assays and specific MMP-2/-9 inhibitor experiments indicated that HMHA1 promotes extracellular matrix degradation by regulating MMP activity, increasing cancer cell invasiveness under hypoxia.

Step Four: HMHA1 Expression and Enhanced Invasiveness Under Post-Radiation Reoxygenation

Simulating post-radiation changes in oxygen levels, researchers found that post-radiation reoxygenation significantly increased HMHA1 expression via the ROS/HIF pathway. Boyden Chamber invasion assays also showed that this treatment significantly enhanced invasiveness in wild-type cells, but not in HMHA1 knockout cells.

Results and Conclusions

Main findings include: 1. HMHA1 expression significantly increases under severe hypoxia, regulated dually by HIF-1 and HIF-2. 2. HMHA1 enhances cancer cell invasiveness primarily through regulating MMP-2/-9 activity and cytoskeleton dynamics. 3. Post-radiation reoxygenation increases HMHA1 expression via the ROS-mediated HIF pathway, significantly enhancing cancer cell invasiveness. 4. TCGA data analysis indicated that cancer patients with high HMHA1 expression have significantly poorer overall survival rates.

Significance of the Study

This research reveals the crucial role of HMHA1 in hypoxic environments of cancer cells and elucidates the molecular mechanism of its expression and increased invasiveness post-radiation. These results provide new perspectives and potential targets for cancer treatment, especially in addressing recurrence and metastasis issues post-radiotherapy. HMHA1 could emerge as a new intervention target for future cancer treatments, and further studies could aid in developing new radiotherapy sensitization strategies, improving clinical outcomes for cancer patients.

Research Highlights

1. HMHA1 expression increases under hypoxia through dual regulation by HIF-1 and HIF-2.
2. HMHA1 enhances cancer cell invasiveness by increasing MMP-2/-9 activity and regulating cytoskeleton dynamics.
3. Post-radiation reoxygenation further increases HMHA1 expression via the ROS/HIF pathway, enhancing cancer cell invasiveness.
4. Cancer patients with high HMHA1 expression have poorer overall survival rates, indicating significant clinical potential for the protein.

Conclusion and Future Directions

This study clearly describes the expression of HMHA1 and its critical role and mechanism in cancer cell invasiveness. These findings advance the understanding of cancer cell behavior in hypoxic and post-radiation reoxygenated environments and raise new scientific questions. For example, whether the role of HMHA1 is consistent in other cancer types, whether there are more unresolved molecular regulatory mechanisms, and what are the prospects for clinical intervention. These will be areas for further exploration in future research.