ROMO1 – a potential immunohistochemical prognostic marker for cancer development

Introduction

Cancer continues to be a leading cause of mortality worldwide, particularly among aging populations. In 2019, there were approximately 23.6 million new cancer cases and 10.0 million cancer-related deaths globally [1]. In 2020, lung cancer ranked as the most common malignancy among men, accounting for 15.4 % of newly diagnosed cases, followed by prostate and colorectal cancers [2]. Among women in 2020, breast cancer took the lead, representing 25.8 % of all new cases, followed by colorectal, lung, and cervical cancers [2].

When it comes to both preventing and treating tumors, the immune system is crucial [3]. Therapies focused on the immune system, such as cancer vaccines, target specific antigens expressed on cancer cell surfaces while sparing healthy cells from drug-related toxicity [4].

Due to tumor diversity and the fact that cancer vaccines target only a limited number of antigens, cancer cells that do not express these antigens or a group of cancer cells with mutations that alter surface antigens can evade immune control and create new tumor populations that can resist treatment with vaccines encoding the same tumor-associated antigens [5, 6]. Therefore, the discovery of new potential biomarkers for cancer development and further treatment is of great importance.

Intratumoral heterogeneity is a hallmark of cancer progression, with malignancies becoming increasingly diverse as the disease advances [7]. Because cancer is a progressive disease, throughout its course, malignancies typically become more diverse. As a result of this heterogeneity, the majority of the tumor may contain a variety of cells with distinct molecular signatures and varying degrees of sensitivity to treatment [7]. Increasing reactive oxygen species (ROS) levels have a significant impact on heterogeneity. Under low concentrations, reactive oxygen species may function as signaling molecules that promote tumorigenesis and heterogeneity, whereas under high concentrations, these species may function as cancer modulators due to their deleterious, genotoxic, or proapoptotic effect on cancer cells [8]. This effect is due to ROS’s capacity to induce genetic and epigenetic alterations in DNA structure [8]. One way to produce ROS in the mitochondria is through the Reactive Oxygen Species Modulator 1 (ROMO1) [9].

Reactive oxygen species modulator 1 – what is it?

Reactive Oxygen Species Modulator 1 (ROMO1), identified in 2006, has been the subject of extensive research regarding its role in ROS production. This protein resides within the inner mitochondrial membrane and generates ROS via Complex III in the electron transport chain. Common ROS types produced include hydrogen peroxide, the hydroxyl radical, and the superoxide anion, generated through the partial reduction of atmospheric oxygen [3].

The structural characteristics of ROMO1 were reported by Lee et al. in 2018 [10]. It consists of two transmembrane domains (TMDs) linked by a basic loop, each containing an alpha helix. TMD2 comprises polar amino acids (K58, T59, Q62, S63, T66, and T69), while TMD1 includes a hydrophobic alpha helix. By forming homo-oligomers in the inner mitochondrial membrane, the amphipathic TMD facilitates the creation of non-selective cation channels in cells. As per Lee et al. [10], ROMO1 functions as a non-selective cation channel with properties akin to viroporins, enabling it to alter the membrane potential and permeability of mitochondrial membranes.

ROMO1 has a notable impact on cancer cell invasion and proliferation, contributing to an ongoing inflammatory response. Multiple studies have examined ROMO1’s role in cancer progression and its potential as a prognostic factor in various cancer types [9]. It has been associated with lymphatic metastasis and a less favorable prognosis [11].

ROMO 1 – methods for examination

ROMO1 can be examined using many methods, however, the most common ones include-RNA sequencing, used to measure gene expression, ELISA and Western blotting, PCR and immunohistochemistry (IHC) (Figure 1).

Figure 1:

Methods for ROMO1 analysis.

The novel protein is a critical factor in cancer, impacting malignancy through its influence on redox balance, mitochondrial dynamics, and apoptosis resistance.

Redox Regulation: ROMO1 generates ROS, which, at high levels due to ROMO1 overactivity, can damage DNA, proteins, and lipids, promoting genetic instability and cancer progression [12]. Mitochondrial Dynamics: ROMO1’s location in mitochondria affects their function and structure [13]. Dysregulation by ROMO1 can lead to energy problems, increased ROS production, and resistance to cell death, contributing to cancer cell survival. Apoptosis Resistance: ROMO1 disrupts the balance of apoptosis-regulating factors within mitochondria, making cancer cells less responsive to apoptotic signals, thereby facilitating their survival and growth despite treatment [14].

A 2022 study by Wang et al. [3] tested the importance of ROMO1 as a biomarker for prostate cancer progression using information from the TCGA data set and various model analyses. This data comes from RNA sequencing. They concluded that ROMO1 is a significant gene strongly linked with the microenvironment of prostate cancer tumors, and the essential signaling pathways implicated were identified. In light of this, they proposed that ROMO1 may serve as a biomarker and therapeutic target for prostate cancer. Another study in 2020 examined ROMO1’s role in bladder cancer and oxidative stress using quantitative real-time PCR [15].

ROMO1 expression was significantly correlated with tumor size and histopathological grading. The expression level of the ROMO1 gene was measured in malignant and adjacent healthy tissues and found to be significantly higher in bladder cancerous tissues than in adjacent healthy tissues [15]. ELISA method and Western blotting were used to measure ROMO1 in patients with non-small cellular lung cancer (NSCLC), where the team tested the potential of ROMO1 as a diagnostic biomarker [16]. They concluded that the serum levels or ROMO1 correlate positively with ROMO1 in cancerous tissues. In addition to that, the serum ROMO1 levels of NSCLC patients were substantially higher than those of healthy individuals or those with benign lung diseases [16]. The different methods of detection of ROMO1 are shown in Table 1.

ROMO1 and immunohistochemistry

Immunohistochemistry (IHC) offers several compelling reasons and advantages for detecting ROMO1 as a potential diagnostic biomarker and therapeutic target in cancer research:

Tissue Localization: IHC allows precise localization of ROMO1 within tissue samples. This spatial information is very important for understanding ROMO1’s role in cancer development and progression. It helps us identify specific cellular and subcellular locations where ROMO1 is overexpressed or dysregulated.

Protein Expression Profiling: ROMO1 protein expression levels in tumor tissues can be evaluated by IHC. The associations between ROMO1 expression and disease severity, prognosis, and therapeutic response can be determined using this information.

Validation of Biomarker Potential: IHC is also a reliable technique for confirming ROMO1’s potential as a diagnostic biomarker. Researchers can evaluate the specificity and sensitivity of ROMO1 as a diagnostic biomarker by contrasting its expression in healthy and malignant tissues.

Patient Stratification: Patient classification based on ROMO1 expression levels can benefit from IHC. By enabling tailored treatment plans, this information can assist clinicians in identifying patients who are more likely to benefit from ROMO1-targeted medicines.

Monitoring Treatment Response: IHC can be used to monitor changes in ROMO1 expression following treatment. Decreased ROMO1 levels post-treatment may indicate therapeutic efficacy, providing a means to assess treatment response.

Clinical Translation: There are several studies that have tested the expression of ROMO1 using IHC and in all of them the results are similar-overexpression of the protein leads to poorer prognosis.

Discussion

Gynecological cancers are a significant public health concern, and early detection through screening is crucial for effective management. Nowadays the only gynecological cancer that is screenable is carcinoma of the cervix (CC) – there is no such option for endometrial, ovarian, and vulvar cancer [31]. Even though a screening programme is available for CC, in 2020 there was a 5.7 % increase in new cases and an 8.9 % increase in morbidity compared to 2018 [32]. That is why new prognostic and predictive biomarkers are required by clinicians.

Bearing in mind that Lee et al. [10] described the structure of ROMO1 very recently – in 2018, this novel protein shows great perspective as a potential biomarker, showing different results according to the type of cancer being studied. The methods by which it can be examined are different, but we decided to use IHC for the following reasons. IHC can be highly specific, as it relies on antibodies that are designed to target a particular protein of interest. This specificity ensures that we are detecting ROMO1 and no other proteins with similar properties. IHC is also very compatible with different tissue samples. It can be used both on paraffin-embedded or frozen sections, allowing for flexibility in sample preparation. This method is suitable for use in clinical practice because it can assess protein expression in patient samples. If ROMO1 is implicated in a particular disease or condition, IHC can be employed for diagnostic or prognostic purposes. Of course, a very alluring reason to use IHC is its price-it is much cheaper than other possible methods, such as RNA sequencing for example.

Different studies [18, 22, 29, 30] using IHC show that ROMO1 expression levels are higher in advanced stages, while in CC it is exactly the opposite – its expression is higher in the early stages of the disease – it may be owing to the viral component in the carcinogenesis of CC.

ROMO1 is responsible for a rise in intracellular ROS levels. We, however, have not tested what role this biomarker plays in the tumor microenvironment. In the study carried out in 2022, the authors concluded that ROMO1 has a high degree of infiltration, a bad prognosis, and a strong connection with CD8+T cells, all of which are associated with tumor immune infiltration [3]. ROMO1-induced ROS accumulation can promote angiogenesis [14], facilitating tumor vascularization and nutrient supply. Additionally, elevated ROS levels may suppress anti-tumor immune responses, allowing cancer cells to evade immune surveillance [33]. Thus, ROMO1’s impact on the tumor microenvironment highlights its potential as a crucial mediator of cancer progression and therapeutic resistance.

Conclusions

ROMO1 regulates redox, mitochondrial dynamics, and apoptosis. Its increasing significance in cancer biology is fascinating and therapeutically significant. It affects ROS levels, mitochondrial dynamics, and the tumor microenvironment. We strongly believe that immunohistochemistry is a promising method to test for ROMO1 expression, as it is inexpensive, accessible, and straightforward to use.

The potential of ROMO1 as both a diagnostic biomarker and therapeutic target opens new avenues for personalized cancer treatment. However, challenges persist. An in-depth understanding of ROMO1’s molecular control and interactions with other cellular components is essential for targeted therapeutics. Clinical translation necessitates the study of ROMO1’s context-dependent effects across various cancer types and patient cohorts. Establishing ROMO1 as a diagnostic biomarker and therapeutic target requires robust preclinical and clinical investigations.