Cancer research has seen a steady shift towards ‘tumor microenvironment’ driven hypotheses. Tumor cells exploit their environment to fuel their own growth and metastases. Thus, targeting the crosstalk between the tumor cells, stromal cells and the immune microenvironment has gained new appreciation for cancer therapeutics.
Paracrine or juxtacrine signaling via soluble chemokines and cytokines are the major known drivers of the crosstalk between the heterotypic cells in a tumor. However, the role of exosomes (extracellular vesicles) as dynamic modulators of intercellular communication has only recently been highlighted. A study published in Cell in July 2017 beautifully unveiled the role of stromal exosomal RNA (exoRNA) as viral mimics driving the tumor-stromal crosstalk in triple negative breast cancer (TNBC). It was co-authored by Cancer Biology student Barzin Nabet from Dr. Andy J. Minn’s lab.
Exosomes are 40-150 nanometer vesicles released by all cell types. They are composed of a lipid bilayer and contain all major cellular constituents – proteins, DNA and RNA. At any given time, the contents of exosomes reflect the phenotypic state of the cell that generates them. The ability of exosomes to shuttle the constituents of one cell to another has added an additional dimension to the complexity of cellular interactions in a tumor microenvironment. In October 2014, another study published by Dr. Minn’s group in Cell highlighted that upon stimulation by tumor cells, stromal cells release exosomes with RNA molecules that have a close sequence similarity to viral RNAs. Inevitably, these RNA molecules are then recognized as damage associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) in the tumor cells, thus initiating a cascade of anti-viral responses. In general, upon interaction with specific PRRs, viral RNAs direct transcriptional activation of interferon stimulated genes (ISGs). Ironically, across many common human cancers, various tumors have been reported to express high levels of ISGs even in the absence of any prior viral infection. Further, studies have shown that patients with tumors expressing high levels of ISGs are more prone to relapse after radiation or chemotherapy. These observations strongly suggest the presence of a viral mimic in the tumor microenvironment.
In the current study, Barzin and colleagues connected the dots to draw out the intricate mechanism by which stromal exoRNAs drive growth and inflammation in the tumor microenvironment. Co-culture experiments between MRC5 human fibroblast and 1833 ISG-responding (ISG-R) breast cancer cell lines as well as RNA sequencing data confirmed the presence of non-coding RNA (ncRNA) transcripts in stromal exosomes. By definition, these ncRNAs do not translate into proteins but function to regulate gene expression at transcriptional and post-transcriptional level. RN7SL1 stood out as the only ncRNA with elevated levels in the exosomes upon co-culture in comparison to the monocultures. Tandem pull-down assays confirmed specific binding of RN7SL1 to RIG-I receptors (PRR) in the tumor cells. This interaction induced ISG expression in both stromal and ISG-R breast cancer cells in a RIG-I dependent manner. Intricate secondary structure at the 5’ end of the RNA stood out as a prerequisite for RIG-I - RN7SL1 binding.
5’-triphosphate (5’-ppp) RN7SL1 is highly abundant in the cell at physiological conditions and thus can bind RIG-I in the cytoplasm at all times. Hence one would wonder why our cells fail to initiate a danger response signal at all times?
“This posed a conceptual glitch to the proposed mechanism,” Barzin shared.
Variable levels of RNA ‘shielding’ by RNA binding proteins (RBPs) was found in cells and exosomes. Normalized minimum free energy calculations revealed markedly shielded RN7SL1 in cells in comparison to highly unshielded RN7SL1 in co-culture exosomes. The extensive shielding by RBPs at physiological conditions prevents RN7SL1’s recognition by RIG-I. However, upon stimulation by tumor cells, stromal cells selectively deploy unshielded RN7SL1 in the exosomes. qRT-PCR and immunoblot analyses after co-culture experiments confirmed the role of signal recognition particle proteins – SRP9 and SRP14 in shielding RN7SL1 from inappropriate recognition by RIG-I in the cytoplasm.
Endogenous RNAs can be extruded into the tumor microenvironment as exosomes. Healthy stromal fibroblasts generate RNAs (green) that are normally shielded by the RNA binding proteins SRP9 and SRP14. However, cancerous fibroblasts increase their expression of a specific endogenous RNA RN7SL (red). Notably, this RNA is not shielded and thus acts as a danger associated molecular pattern (DAMP) similar to viral RNA. The presence of this DAMP can be detected by the intracellular pattern recognition receptor RIG-I. Stimulating RIG-I in tumor cells induces interferon stimulated genes (ISGs), which ultimately promotes cell growth and metastasis.
Transcriptional profiling of stromal and tumor cells upon co-culture revealed the activation of NOTCH juxtacrine signaling pathway. This fits well with the requirement of physical cell-cell interaction for ISG induction. Transcription factor MYC being downstream of NOTCH signaling, was also upregulated and its overexpression drove RNA Polymerase (POL 3) and RN7SL levels in the stromal cells. Exosomes derived from MYC-activated stromal cells were injected in athymic nude mice carrying flank tumors. A substantial increase in myeloid cell infiltration with cells displaying high levels of activation markers like CD40, CD86, PDL1 and MHC-II was seen. The impact on T cell infiltration levels, however, is yet to be studied.
Barzin and colleagues demonstrated increased tumorigenic and metastatic potential of tumors expressing high RIG-I signaling and ISG levels through RIG-I KO studies in mice. Finally, they collected serum samples from two cohorts of cancer patients and performed exoRNA-sequencing analysis. The results of these studies confirmed an expected increase in unshielded RN7SL1 and POL3 transcripts levels in serum samples from the cancer patients compared to healthy controls.
Horizontal transfer of DAMPs is a shared feature between viral infections and tumor-stromal cell interactions. Hence, the current study presents a classic case of viral mimicry where the stromal virions crosstalk with tumor cells thus activating anti-viral signaling in the microenvironment. This has provided new insights for directing research towards exosomal crosstalk as a potential target for cancer therapeutics.
Nabet, B.Y., Qiu, Y., Shabason, J.E., Wu, T.J., Yoon, T., Kim, B.C., Benci, J.L., DeMichele, A.M., Tchou, J., Marcotrigiano, J., Minn, A.J. Exosome RNA Unshielding Couples Stromal Activation to Pattern Recognition Receptor Signaling in Cancer. Cell 2017; 170(2):352-366.e13.