By Kate Haley, Post-Doc, RCSI
At present, the field of biomedical science is replete with researchers endeavoring to unveil molecular mechanisms which will improve our understanding of disease pathogenesis and in turn be translated into the clinic as therapeutic strategies. The advent of translational research, nearly 10 years ago now, was marked by a call for increased attention to the interface of basic science and clinical medicine. Perhaps lost in translation, knowledge accrued in the basic sciences was not following an efficient path to the clinic. To improve advances in prevention, diagnosis, and treatment of disease, the NIH established the National Center for Advancing Translational Sciences in 2011. Translational research, as a two-stage model, was first defined by T1 bench-to-bedside research, and T2 research wherein findings from clinical practice were translated to every day practice (Wolf, 2008). Today, nearly a decade later, biomedical research is dominated by a translational-driven approach. While the advances resulting from translational research are innumerable, the ‘translational’ buzzword gives us little opportunity to stop and question whether the path from the bench to bedside has truly reached the height of efficiency. As scientists endeavoring to better understand resistance mechanisms or druggable pathway targets, we pose questions on a daily basis. But instead of focusing on the pathways and the targets, should we be questioning the path?
Clinical Data: The First Step
Take for example, studying endocrine therapy resistance in invasive lobular carcinoma (ILC). ILC is the second most common type of breast cancer, accounting for approximately 10% of all breast tumours. The majority of ILC breast cancers are treated with endocrine therapy such as tamoxifen or fulvestrant, however, one in three women are resistant to endocrine therapy. The absence or lower quality of treatment options for chemoresistant patients highlights a clear demand for translational research in the field. In determining what is causing chemoresistance in these patients, it is logical to go straight to the source: patient samples. In the case of ILC, RNA sequencing was performed on 61 primary samples from the RATHER cohort, and the findings indicated that high expression of the epigenetic reader, bromodomain protein 3 (BRD3) is associated with poor recurrence free survival. While analysis of primary samples provides an accurate indication of pathways or genes which are correlated with poor clinical outcome, tracing back upstream to determine how dysregulated signaling pathways are regulated at the genetic or epigenetic level is a far more complex feat.
Bedside to Benchwork: Finding the Path
Data generated from primary clinical samples often presents multiple paths to pursue when investigating the mechanism underlying drug resistance or a clinical phenotype. When several pathways are upregulated or downregulated, the experimental approach often comes down to a process of elimination. In the case of ILC, when high BRD3 expression was found to be correlated with poor recurrence free-survival, the next logical experiment was to test ILC cell lines for sensitivity to JQ1, an inhibitor of BET family proteins in vitro.
Our findings suggest that ILC cell lines are more sensitive to JQ1 compared to endocrine therapy in vitro. Using an MTT viability assay, we further investigated whether the combination of JQ1 and endocrine therapy is synergistic in ER positive ILC cell lines. Combination of both JQ1 and tamoxifen as well as JQ1 and fulvestrant were found to be synergistic in ER positive cell lines. Finally, we performed paired-end RNA sequencing in an ILC cell line that was sensitive to JQ-1 induced apoptosis and in an ILC cell line that was resistant to JQ1 induced apoptosis in order to identify potential factors contributing to JQ1-induced apoptotic resistance. Gene ontology analysis using DAVID identified 6 pathways upregulated in the JQ1 resistant ILC cell line that were not upregulated in the JQ1 sensitive ILC cell line, including MAPK signaling, Wnt signaling, and insulin resistance. qPCR validation of our RNA sequencing data further suggests that ILC cell lines that are resistant to JQ1-induced apoptosis may be dependent on sustained or high expression of anti-apoptotic BCL-XL after JQ1 treatment. Based on these findings, we plan to further validate the upregulated genes in the JQ1 resistant cell line, including the FGFR signaling pathway. This data will inform our understanding of how BRD3 may play a role in chemoresistant ILC patients, and may potentially serve as a novel therapeutic target in vivo.
Reassessing the experimental path: Is it efficient?
We pursue each pathway with due diligence and eventually elucidate the mechanism. As a logical progression, in vitro findings are then translated to in vivo experiments, which may eventually evolve into Phase I trials. However, when the end outcome of in vitro research is undeniably translational, it is often uncommon that we stop to question how accurate or effective this in vitro work is. Gillet et al. broached this question around the advent of translational research in a NIH publication titled “Redefining the relevance of established cancer cell lines to the study of mechanisms of anti-cancer drug resistance” (Gillet et al., 2011). The authors acknowledge in vitro models as a cornerstone of cancer research and drug development, but question the direct applicability of this research to clinical practice. The authors express concern at their findings which demonstrate that all cancer cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of tissue original, than to clinical samples (Gillet et al., 2011). The authors emphasize the necessity of finding better in vitro or in vivo models which more closely resemble cancer microenvironment and gene expression profiles in vivo.
Finding new translational approaches
So what are the other options then, you might ask? ILC is a surprisingly pertinent example for this Achilles heel of in vitro experimental approaches insofar as there are a limited number of ILC cell lines available for in vitro research in this field. The challenge is thus finding alternative models with which to study the mechanisms underlying drug-resistance. I am most excited by this project when it requires creativity of experimental approach, such as the used of organoids, 3D culture, and PDX models in vivo. While in vitro work is still an undeniable cornerstone of cancer research, we may benefit from challenging ourselves to always assess the translational impact or efficacy of using in vitro models when alternative methodologies may be available. The full circle translational path from primary patient samples, to in vitro work, to mouse models, and back to the clinic again may be long and arduous, but we should seek out more effective experimental paths where possible.
Woolf SH. The Meaning of Translational Research and Why It Matters. JAMA. 2008;299(2):
Gillet J-P, Calcagno AM, Varma S, et al. Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(46):18708-18713. doi:10.1073/pnas.1111840108