Exploring the Dark Proteome: A Hidden Frontier in Cancer Research

Proteins shape tumor behavior. 

Recently, the discovery of the “dark proteome” has highlighted that our current methods of studying proteins may miss a key part of the proteome, indicating that a previously overlooked group of proteins could be influencing tumor biology. 

“The dark proteome refers to a collection of undiscovered and uncharacterized protein types that are produced from alternative genomic loci to known coding sequences or due to RNA mistranslation,” Prof. Reuven Agami, leader of the Division of Oncogenomics at the Netherlands Cancer Institute, told Technology Networks. 

Cancer cells have been found to express proteins from the dark proteome—including proteins that have amino acid alterations without a corresponding DNA mutation, microproteins that stem from historically non-coding regions of the genome, and stress-induced aberrant proteins. 

Agami leads the Cancer Grand Challenges team ILLUMINE, which aims to identify where these dark proteome products come from, how they influence cancer cell behavior, and whether they can be used as therapeutic targets.

 

Technology Networks spoke with Agami to learn more about team ILLUMINE’s work, how the dark proteome influences cancer biology, and how it could inform the discovery of new therapeutics.

The dark proteome in cancer research 

Understanding the dark proteome may reveal new aspects of cancer pathogenesis. Dark proteins are varied and have unique characteristics, which pose both challenges and opportunities. 

“The challenge is to effectively identify [the dark proteins], select the ones that are cancer-specific and shared by many tumors, and engineer novel ways to target them with cancer therapy,” Agami said. 

Microproteins 

Microproteins (also called miniproteins, micropeptides, or peptideins) from non-canonical open reading frames can modulate the activity of protein complexes, alter cell function by acting as inhibitors or activators of key enzymes, and regulate translation and alternative splicing of mRNA transcripts. 

Through these roles, some microproteins may contribute to tumor growth, metastasis, and drug resistance, whereas others may exert anti-tumor effects. 

The small size of the microproteins also allows them to exit the cancer cell, which “potentially influences the function of other cells in the tumor microenvironment in ways we cannot foresee today,” Agami explained. 

Mistranslated polypeptides 

Mistranslated polypeptides are proteins that are created when ribosomes incorporate the wrong amino acid during translation. These peptides are distinct from genetically encoded mutants. For example, when tryptophan is depleted, protein synthesis continues with phenylalanine in its place, creating W>F “substitutant” peptides.  

Agami and his colleagues found that W>F substitutants are highly abundant in multiple cancer types and are associated with increased oncogenic signaling. While W>F substitutants can impair protein activity, they can also alter antigen presentation at the cell surface, which leads to T cell recognition, activation, and cancer cell killing. 

Dysregulated mRNA translation signaling in cancer cells can also alter protein production and create dark proteins, allowing the cells to survive the harsh conditions of the tumor microenvironment. This leads to the formation and presentation of neoepitopes—cancer-specific peptides—at the cell surface, which could be immunologically targeted.  

“Targeting cancer-specific dark proteins can provide new ways to tackle resistance to current therapies” — Prof. Reuven Agami. 

Retroviral proteins 

Remnants of ancient retroviral infections are fixed in the human genome. While these genes are typically silenced in healthy cells, evidence suggests that they can be expressed in cancers. The proteins produced from these retroviral genes form part of the dark proteome. 

A causal role for retroviral proteins in cancers hasn’t been established, but studies indicate that human endogenous retroviral (HERV)-K protein is associated with increased migration, proliferation, and cancer cell survival. An increase in HERV-W protein expression via a recurrent mutation in the 3’ UTR region has been observed in bladder cancers, suggesting that HERV expression may promote cancer progression. 

As HERV proteins are largely present only in cancer cells, they are an attractive therapeutic target. 

Degraded proteolytic peptides 

Substrate degradation by the proteasome is a critical regulator of cell function. The peptides formed by proteasomal cleavage are usually further processed into amino acids, but a subset of these peptides evade this step and play key roles in cell signaling, antigen presentation, and apoptosis. 

The identification and characterization of these degraded peptides is challenging due to their diversity, hence their inclusion in the dark proteome. 

In cancer cells, proteosome activity can become dysfunctional, which supports cell growth and survival and results in a different landscape of degraded peptides.  

This alteration in proteasomal cleavage patterns can influence antigen diversity and impede the detection and destruction of tumor cells by the immune system. 

Creating a dark proteome atlas 

The absence of comprehensive reference catalogs for the dark proteome—in both normal and cancerous cells—hinders progress in understanding the cancer dark proteome. 

Furthermore, the stresses placed on cancer cells, such as exposure to chemotherapy agents, nutrient deprivation, and metabolic stress, can spur dark protein generation. However, there are currently only limited insights into these mechanisms. 

ILLUMINE is therefore aiming to create an atlas of the dark proteome in cancer, focusing on cancers that are challenging to treat. 

“We will focus on the five most difficult-to-treat cancer types, including pancreatic, brain, ovarian, lung, and leukemia cancer types,” said Agami. “In those tumor types, we will take a multiomics approach and will subject cancer models to cancer-induced cell stress to uncover cancer dark proteins.” 

As the cancer dark proteome is mapped, Agami and his team will identify any recurring events that contribute to the formation of dark proteins. From there, they plan to study the molecular mechanisms behind cancer-specific dark protein expression and identify links to oncogenic pathways. 

The team will also “design screens to identify drugs to be used for cancer treatment,” Agami said. 

“Depending on the type of cancer dark protein, targeting them involves designing immunotherapies (mRNA vaccination and adaptive T cell therapy), peptide therapy, or drug therapy to stimulate or inhibit their expression in case of oncogenic and tumor-suppressive functions,” he explained. 

Challenges in studying the dark proteome 

The dark proteome has been likened to dark matter in physics, in that it is not directly visible with standard tools. Dark proteins have no experimentally resolved structures, lack structural homologs, and cannot be predicted by computational approaches. 

However, Agami highlighted that tools to detect dark proteins do exist. “It includes ribosome profiling to identify translated regions, proteomics, and immunopeptidomics,” he said. 

“The challenge is to identify cancer-specific dark proteins and antigens” — Prof. Reuven Agami. 

One of the challenges facing the ILLUMINE project is that some dark proteins may not be cancer-specific, meaning they can’t be used as a therapeutic target. Other dark proteins might be expressed only at very low levels, making them difficult to use for therapies. 

Changing current paradigms in cancer research 

Insights from the dark proteome have the potential to shift existing paradigms in cancer research, particularly in drug discovery and biomarker development. 

“The unique characteristics of the dark proteins may open new avenues in cancer therapy.” — Prof. Reuven Agami 

“Understanding the oncogenic link of dark proteins may identify vulnerabilities that can be exploited for drug therapies,” explained Agami.  

He also highlighted that if we can master their targeting, stress-induced dark antigens could provide additional cancer-specific drug targets. 

Furthermore, “the expression of certain microproteins or stress-induced mistranslation could be developed as a biomarker for cancer aggressiveness and personalized therapeutic approaches,” Agami noted.