Viral hijacking of the Hippo pathway - HCMV-encoded viral GPCRs US28 and UL78 differentially modulate the Hippo pathway in cancer.

Project Information

ViHiHippo

Grant agreement ID: 101063969

DOI

10.3030/101063969

Project closed

EC signature date 9 June 2022

Start date 1 July 2022

End date 31 October 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 187 624,32

Coordinated by

STICHTING VU
Netherlands

Periodic Reporting for period 1 - ViHiHippo (Viral hijacking of the Hippo pathway - HCMV-encoded viral GPCRs US28 and UL78 differentially modulate the Hippo pathway in cancer.)

Reporting period: 2022-07-01 to 2024-10-31

Summary of the context and overall objectives of the project

The ViHiHippo project aimed to address the critical issue of human cytomegalovirus (HCMV) and its potential role in cancer progression. HCMV is a herpesvirus with a high prevalence, infecting over 60% of the European population. While HCMV typically causes minimal symptoms in healthy individuals, recent studies have suggested that it may contribute to serious diseases such as heart disease and cancer. Notably, HCMV has been detected in tumor tissues but not in surrounding healthy tissues, and its presence is associated with increased tumor growth and poorer clinical outcomes in cancer patients. This underscores the urgent need to understand how HCMV influences cancer progression and to develop effective treatments targeting the latent HCMV pool.
The primary objective of the ViHiHippo project was to elucidate the molecular mechanisms by which HCMV-encoded viral G protein-coupled receptors (vGPCRs), specifically US28 and UL78, modulate the Hippo signaling pathway. The Hippo pathway is crucial for regulating cell proliferation and organ growth, and its dysregulation is linked to cancer. Understanding how these vGPCRs hijack the Hippo pathway provided critical insights into HCMV's potential oncomodulatory effects.
The project was structured to achieve the following specific objectives:
1. Identify and Characterize US28 Signaling: Investigate the signaling cascade that leads to US28-mediated regulation of the Hippo pathway, providing a foundational understanding of how US28 influences cell proliferation and tumor growth.
2. Investigate UL78 Signaling: Explore the signaling mechanisms of UL78, an atypical GPCR that does not activate classical G proteins, to uncover novel signaling routes through which UL78 modulates the Hippo pathway.
3. Characterize Combined Effects in Cancer: Examine the outcomes of US28 and UL78 modulation of the Hippo pathway in a viral cancer setting, including testing for potential complementary effects when both vGPCRs are co-expressed, to provide insights into their collective impact on cancer progression.
The ViHiHippo project aligned with the European Union's strategic priorities in health research, particularly in understanding and combating cancer. By addressing the role of HCMV in cancer, this research contributed to the broader goals of reducing cancer incidence and improving patient outcomes. The project's significance was underscored by the high prevalence of HCMV and its potential impact on cancer progression. The results provided new therapeutic targets and strategies for managing HCMV-associated cancers, thereby supporting the EU's efforts to enhance public health and healthcare innovation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The ViHiHippo project aimed to uncover how human cytomegalovirus (HCMV) proteins, specifically the viral G protein-coupled receptors (vGPCRs) US28 and UL78, affect the Hippo signaling pathway, which is important for controlling cell growth and organ size. The project was divided into three main objectives:
Objective 1: Identify and Characterize US28 Signaling
To understand how US28 affects the Hippo pathway, we used HEK293 cells, which are commonly used in lab experiments. These cells were modified to produce US28 and a reporter that shows activity in the Hippo pathway. We used gene-editing techniques and various inhibitors to identify which proteins are involved in US28's effects.
Key achievements:
• Discovered that US28's constant activity (without external signals) drives changes in the Hippo pathway, while activation by chemokines (signaling molecules) does not.
• Found that US28 signals through a specific group of proteins called the Gq family to influence the Hippo pathway.
• Determined that other proteins, including G12/13, beta-arrestins, and PLCβ1-4 enzymes, are not necessary for US28's effects on the Hippo pathway.
• Confirmed that a protein called YAP is essential for US28 to affect the Hippo pathway.
Objective 2: Investigate UL78 Signaling
Unlike US28, UL78 does not use the usual signaling proteins. We created various modified versions of UL78 to find out which parts of the protein are important for its function. We also used gene-editing and inhibitors to study its signaling.
Key achievements:
• Found that a specific part of UL78, called the DRY motif, is not needed for its signaling, but a cluster of serine and threonine amino acids at the end of the protein is crucial.
• Determined that the usual signaling proteins, including G proteins and beta-arrestins, are not involved in UL78's effects on the Hippo pathway.
• Developed nanobodies (small antibody fragments) that bind to UL78 and can change its activity.
Objective 3: Characterize Effects in Cancer
To see how our findings apply to a more realistic setting, we studied the effects of US28 and UL78 in U251 glioblastoma cells, a type of brain cancer cell. We used systems that allow us to control the production of US28 and UL78 in these cells and measured the activity of the Hippo pathway.
Key achievements:
• Confirmed that US28 affects the Hippo pathway in U251 glioblastoma cells.
• Found that US28 increases the expression of a marker called CD133, which is associated with cancer stem cells, in U251 cells.
• Observed that US28-expressing U251 cells show changes in shape that indicate a lack of differentiation (maturation into specialized cells).
• Found evidence that US28 may promote stem cell-like properties in glioblastoma cells, including increased CD133 expression and lack of differentiation.
• Obtained inconclusive results for UL78's effects in U251 cells, possibly due to low levels of UL78 production.
Overall, the ViHiHippo project provided important insights into how HCMV proteins US28 and UL78 affect the Hippo signaling pathway and their potential roles in cancer progression. These findings could help develop new treatments for HCMV-associated cancers.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The ViHiHippo project has yielded several results that advance our understanding of how human cytomegalovirus (HCMV) proteins US28 and UL78 modulate the Hippo signaling pathway, with significant implications for cancer research and treatment.
Overview of Results
1. US28 Signaling Mechanism: We identified that US28 signals through the Gq family of G proteins to modulate the Hippo pathway, a novel finding that highlights the unique signaling properties of this viral receptor. This discovery provides a new perspective on how HCMV can influence cell proliferation and tumor growth.
2. UL78 Signaling Mechanism: Our research revealed that UL78 modulates the Hippo pathway through a non-canonical signaling mechanism, independent of classical G proteins and beta-arrestins. This finding opens up new avenues for understanding atypical GPCR signaling and its role in viral pathogenesis.
3. Cancer Cell Impact: We demonstrated that US28 promotes stem cell-like properties in glioblastoma cells, including increased expression of the stemness marker CD133 and inhibition of differentiation. These results suggest that HCMV may contribute to cancer progression by maintaining a population of cancer stem cells.
4. Nanobody Development: We developed nanobodies that can bind to UL78 and modulate its activity, providing a potential therapeutic tool for targeting HCMV-infected cells.
Potential Impacts and Key Needs
The results of the ViHiHippo project have several potential impacts and highlight key needs for further uptake and success:
1. Further Research: Additional studies are needed to fully elucidate the signaling pathways and molecular mechanisms by which US28 and UL78 modulate the Hippo pathway. This includes exploring the downstream effects of these interactions and their implications for cancer progression.
2. Therapeutic Development: The development of nanobodies targeting UL78 represents a promising therapeutic approach. Additionally, our research supports the potential of previously published US28-targeting nanobodies as a useful treatment strategy for HCMV-dependent oncomodulation and other HCMV-dependent diseases and effects. Further research and development are required to optimize these nanobodies for clinical use, including testing their efficacy and safety in preclinical and clinical trials.
3. Fundamental Research Foundation: This project was focused on fundamental research and has laid the groundwork for a number of steps in areas such as commercialization, access to markets, finance, and regulatory support. However, at this stage, we cannot define these steps clearly. Future research and development will be necessary to translate these foundational findings into practical applications.
Overall, the ViHiHippo project has provided valuable insights into the role of HCMV in cancer and identified potential therapeutic targets. Continued research and development, supported by a robust framework for commercialization and regulation, will be key to realizing the full potential of these findings.