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Updated: Dec 14, 2025
Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
RhoB is regulated by hypoxia and modulates metastasis in breast cancer
Julia A Ju1,2,3, Inês Godet1,3, Josh W DiGiacomo1,3
1Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Background:
RhoB is a Rho family GTPase that is highly homologous to RhoA and RhoC. RhoA and RhoC have been shown to promote tumor progression in many cancer types; however, a distinct role for RhoB in cancer has not been delineated. Additionally, several well-characterized studies have shown that small GTPases such as RhoA, Rac1, and Cdc42 are induced in vitro under hypoxia, but whether and how hypoxia regulates RhoB in breast cancer remains elusive.
Aims:
To determine whether and how hypoxia regulates RhoB expression and to understand the role of RhoB in breast cancer metastasis.
Methods:
We investigated the effects of hypoxia on the expression and activation of RhoB using real-time quantitative polymerase chain reaction and western blotting. We also examined the significance of both decreased and increased RhoB expression in breast cancer using CRISPR depletion of RhoB or a vector overexpressing RhoB in 3D in vitro migration models and in an in vivo mouse model.
Results:
We found that hypoxia significantly upregulated RhoB mRNA and protein expression resulting in increased levels of activated RhoB. Both loss of RhoB and gain of RhoB expression led to reduced migration in a 3D collagen matrix and invasion within a multicellular 3D spheroid. We showed that neither the reduction nor overexpression of RhoB affected tumor growth in vivo. While the loss of RhoB had no effect on metastasis, RhoB overexpression led to decreased metastasis to the lungs, liver, and lymph nodes of mice.
Conclusion:
Our results suggest that RhoB may have an important role in suppressing breast cancer metastasis.
Insights
Hypoxia upregulates RhoB, a GTPase, in breast cancer. While RhoB doesn't affect tumor growth, its overexpression suppresses metastasis, suggesting a role in preventing cancer spread.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Signaling
Background:
- RhoB is a Rho family GTPase homologous to RhoA and RhoC.
- The role of RhoB in cancer, particularly breast cancer metastasis under hypoxia, is not well understood.
- Hypoxia's regulation of small GTPases like RhoA, Rac1, and Cdc42 is known, but its effect on RhoB remains elusive.
Purpose of the Study:
- To investigate how hypoxia influences RhoB expression and activation in breast cancer.
- To elucidate the specific role of RhoB in breast cancer metastasis.
Main Methods:
- Quantitative PCR and Western blotting were used to assess RhoB expression and activation under hypoxia.
- CRISPR-mediated RhoB depletion and RhoB overexpression models were employed.
- 3D in vitro migration and invasion assays, along with an in vivo mouse model, were utilized to evaluate RhoB's functional significance.
Main Results:
- Hypoxia significantly increased RhoB mRNA and protein levels, leading to higher activated RhoB.
- Both RhoB depletion and overexpression reduced cell migration and invasion in 3D models.
- Tumor growth in vivo was not affected by altered RhoB levels.
- RhoB overexpression, but not its loss, decreased metastasis to lungs, liver, and lymph nodes.
Conclusions:
- RhoB expression is upregulated by hypoxia in breast cancer.
- RhoB plays a significant role in suppressing breast cancer metastasis.
- Targeting RhoB may offer a therapeutic strategy to inhibit cancer spread.
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