Suppressing breast cancer metastasis: The role of hypoxia-induced RhoB expression and activation

By Jamshed Arslan, Pharm. D., PhD.

The Ras homologous (Rho) GTPase family of signaling molecules has over 20 members, which typically cycle between active (GTP-bound) and inactive (GDP-bound) states. These small GTPases primarily act through cytoskeleton to regulate cell/tissue morphology and function. The Rho subfamily within the Rho GTPase family comprises three highly identical proteins: RhoA, RhoB and RhoC. When compared with RhoA and RhoC, RhoB is the most versatile member of the subfamily and it is not an essential gene in mice. While RhoA and RhoC are induced by hypoxia to promote cancer, the effect of hypoxia on RhoB and its potential role in cancer remain inconclusive. To solve these unknowns, scientists from the University of Maryland    and Johns Hopkins University   , USA, employed both in vitro and in vivo models. They used breast cancer cells, 3D migration models and a mouse model to discover that hypoxia upregulates RhoB mRNA and protein levels, and that RhoB overexpression decreases breast cancer metastasis.

Metastasis suppressors prevent cancer growth in secondary tissues. Molecules that inhibit cancer metastasis may act through a range of mechanisms (e.g., changes in cell adhesion and modulation of signaling pathways relevant for cell motility and survival)

Hypoxia induces RhoB expression and activation

To determine the effect of hypoxia on RhoB, the team exposed several breast cancer cell lines to hypoxic (1% O2) or normoxic (20% O2) culture conditions. Quantitative PCR revealed up to 6-fold increased expression of RhoB gene in hypoxic conditions. The immunoblots of the cell lysates and RhoB-GTP pull down assay showed hypoxia-driven RhoB protein expression and activation. The researchers then moved on to determine the effect of RhoB expression itself (under normoxia) on cell motility and invasive capacity.

Loss or gain of RhoB decreases cell motility and invasion in 3D migration models

Since RhoB’s effect on cell motility and invasion might provide a clue about tumor progression and metastasis, the researchers embedded subclones with or without RhoB into 3D collagen matrix and tracked cell movements. Interestingly, both the CRISPR gRNA-induced abrogation and vector overexpressing RhoB led to decreased 3D cell motility. Likewise, live-cell microscopy of breast cancer spheroids revealed that RhoB loss or gain decreased invasion distance.

At this point in the study, the researchers concluded that hypoxia induces RhoB, but a change in RhoB expression itself decreases cell motility and invasion, as seen in 3D migration models under normoxia. They wanted to understand the effect of the hypoxia-RhoB interplay on tumor growth and metastasis in mammals.

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RhoB expression does not affect tumor growth but RhoB overexpression decreases breast cancer metastasis

To determine the effect of RhoB expression on tumor growth, the team injected immunodeficient mice with RhoB knockdown or control breast cancer cells. In these two groups, image analysis revealed a similar tumor size, and Haemotoxylin and Eosin (H&E) staining indicated no difference in lung metastasis. After recording the results of RhoB knockdown, the researchers injected different immunodeficient mice with RhoB overexpressing breast cancer subclones. Again, tumor growth did not differ between control and test groups. However, H&E staining and qPCR of HK2 gene to quantify human DNA content revealed a decreased metastasis towards lungs or livers in mice with RhoB-overexpressing tumors.

Coupled with the observation that high levels of RhoB increase the overall survival of breast cancer patients, the researchers’ final conclusion was that RhoB is associated with improved prognosis.

Significance of RhoB as a potential therapeutic target

Apart from shedding light on the hypoxia-RhoB interplay in breast cancer, this study raises interesting research questions. For example: is HIF-1 alpha/HIF-2 alpha required in hypoxia-driven RhoB expression/activation? What are the downstream pathways of diminished metastasis due to hypoxia induced RhoB expression/activation? What levels of RhoB can significantly inhibit metastasis? The answers to these questions can promote the use of RhoB to suppress metastasis in light of the current study.

Jamshed Arslan, Pharm D, PhD   
Dr. Arslan is an Assistant Professor at Dow University of Health Sciences, Pakistan,
where he teaches Pharmacology to future pharmacists.

References

Ju, J. A., Godet, I., DiGiacomo, J. W., & Gilkes, D. M. (2019). RhoB is regulated by hypoxia and modulates metastasis in breast cancer. Cancer Reports. https://doi.org/10.1002/cnr2.1164