Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer, ranking as the fourth leading cause of cancer-related deaths in the world [1]. With a typical overall survival of 6 months from diagnosis, developing sensitive diagnostic tools and treatments is critical for managing this disease [2]. Current treatment options are complicated by the broad heterogeneity in genetic mutations observed in PDAC tumors, which frequently renders the tumors chemo-resistant [1]. Standard treatments remain largely palliative, aiming to prolong survival and ease symptoms; and conventional cytotoxic treatments such as chemotherapy and radiation therapy remain marginally beneficial. Thus, an active area of research seeks to identify novel, targeted therapies that effectively treat PDAC.
A recent study published by Dr. Todd Ridky’s group in Cellular and Molecular Gastroenterology and Hepatology provides important insight into the efficacy of a putative PDAC therapy. Led by first-author Dr. Christopher Natale, a recent DSRB graduate, this work builds off of the frequent observation that female sex is associated with lower incidence and improved clinical outcomes for many cancers. Thus, biological differences between sexes, for example estrogen hormone signaling in women, likely influence tumor development, progression, and therapeutic response. Understanding the mechanisms that underlie the observed protective effect of female sex may be critical to identifying new cancer therapeutics.
Natale and colleagues hypothesized that nonclassical estrogen signaling mediated through the G protein-coupled estrogen receptor (GPER) may be key to understanding the sex differences observed in cancer etiology. Previous work conducted by Dr. Ridky’s group revealed that stimulation of GPER, which is expressed in a wide number of tissues including melanocytes, inhibits tumor growth in cutaneous melanomas [3]. Importantly, melanocytes do not express classical estrogen and progesterone nuclear hormone receptors, suggesting that GPER activation alone drives an antitumor response. GPER activation also has tumor suppressive properties in multiple other cancers, such as colorectal cancer and non-small cell lung cancer [4,5]. In the present study, Natale et al expands upon these findings to determine whether GPER signaling is therapeutically beneficial in PDAC tumors.
To investigate GPER and its contribution to PDAC, Natale and colleagues utilized murine PDAC tumor lines as their model system. Recently developed by Ben Stanger’s lab at Penn, these tumor lines represent different degrees of immune cell infiltration and responsiveness to cytotoxic and immune therapies. The cell lines ranged from minimal CD8+ T-cell infiltration with poor response to therapy (6419c5 cell line) to robust CD8+ T-cell infiltration with modest (6499c4 cell line) and strong (2838c3 cell line) therapy responsiveness. Thus, these cell lines represent the broad heterogeneity often observed in PDAC tumors.
The authors first asked how PDAC tumor cells respond to GPER activation. After treating each tumor cell line with G-1, a highly specific small molecule GPER agonist, the authors measured cell proliferation, as well as protein and gene expression changes. Strikingly, they observed a significant reduction in proliferative cells in each cell line, with an associated block to the G1-S phase transition of the cell cycle. Furthermore, a key oncogene, c-Myc, was shown to be downregulated following G-1 treatment. RNA-Seq profiling of gene expression changes induced following G-1 treatment further confirmed the downregulation of gene sets related to cell proliferation and invasion. Together, these results suggest that activation of GPER signaling reduces tumorigenicity in mouse models of PDAC.
Translating these findings into an in vivo model, Natale and colleagues next injected PDAC tumor cells into the subcutaneous flanks of mice, thereby inducing tumor growth. A subset of these tumor-bearing mice were then treated with G-1, which resulted in reduced tumor volumes and prolonged animal survival, supporting a tumor suppressive effect of GPER signaling in vivo. Importantly, both male and female mice harboring subcutaneous PDAC tumors responded similarly to G-1 treatment, suggesting that the antitumor activity of GPER signaling is not dependent on sex.
An important sub-field of cancer treatments utilizes immunotherapies to induce an anti-tumor immune response that targets tumor cells. Importantly, inhibition of the immune checkpoint programmed death 1 (PD-1) receptor using a monoclonal antibody (αPD-1) has been shown to prevent T-cell inhibition, promote anti-tumor immune responses, and reduce tumor growth [6]. Recognizing the critical function of immunotherapies, Natale and colleagues next asked whether G-1 treatment can act synergistically with αPD-1 therapy to reduce tumor burden. PDAC-bearing mice treated both with G-1 and αPD-1 demonstrated lower tumor volumes and prolonged survival when compared to mice treated with G-1 or αPD-1 alone. This combinatorial effect was only observed in mice with tumors derived from PDAC cell lines that express the αPD-1 ligand, PD-L1. Together, these results reveal that G-1 treatment increases the efficacy of αPD-1 immunotherapy and may provide a viable combinatorial treatment option for PDAC tumors that express the PD-1 ligand.
In a final set of experiments, Natale and colleagues sought to determine the function of GPER signaling in human PDAC. The authors first demonstrated that GPER is expressed to varying levels in clinical PDAC tissues, with approximately 61% of tested samples showing GPER expression. Next, treatment of three different human PDAC tumor lines with G-1 suppressed cell proliferation and reduced c-Myc protein expression. Furthermore, mice harboring subcutaneous human PDAC tumors responded to G-1 treatment with prolonged survival and tumor regression, demonstrating a tumor suppressive function of GPER activation in human PDAC cases.
Together, the study presented by Natale and colleagues identifies and validates a tumor suppressive effect of GPER signaling in pancreatic ductal adenocarcinoma. Both murine and human PDAC cell lines responded positively to treatment with G-1, a potent and highly selective agonist of GPER; and in vivo studies advanced these findings to reveal G-1-mediated suppression of PDAC tumorigenicity in a clinically-relevant model.
When asked about the overall implications of his study, Dr. Natale notes that “given the anticancer activity observed in PDAC, melanoma, and other cancer models, developing GPER agonists could be a useful therapy in multiple malignancies.” Indeed, the expression of GPER in other tissues suggests that G-1 may act in a tumor suppressive manner in other cancers that are not typically considered sex hormone responsive, such as PDAC. Even more, significant sex differences were not observed following G-1 treatment, suggesting that both men and women will benefit from GPER agonist therapies in the future. Together with the observation that G-1 enhances the response of PDAC tumors to immunotherapy, this study identifies G-1 as a promising putative therapeutic for cancer patients.
Since publishing, Dr. Natale’s and Dr. Ridky’s science has continued to advance. When asked about their next steps, Dr. Natale shares that they have been “focusing on identifying predictive biomarkers and the most sensitive cancer types.” In an exciting new venture, Dr. Natale and Dr. Ridky teamed up with the Penn Center for Innovation to establish Linnaeus Therapeutics, a start-up company designed to “advance these discoveries to clinical trials.” To learn more about their company’s mission and to follow their progress towards developing GPER agonist therapies, visit the Linnaeus Therapeutics website at https://linnaeustx.com/.
References:
1. Aleksandra Adamska, Alice Domenichini, and Marco Falasca. June 2017. Pancreatic Ductal Adenocarcinoma: Current and Evolving Therapies. International Journal of Molecular Sciences. 18(7):1338.
2. Eric A Collissonn, Anguraj Sadanandam, Peter Olson, William J Gibb, Morgan Truitt, et al. April 2011. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nature Medicine. 17(4):500-503.
3. Christopher A Natale, Jinyang Li, Junqian Zhang, Ankit Dahal, Tzvete Dentchev, Ben Z Stanger, Todd W Ridky. January 2018. Activation of G protein-coupled estrogen receptor signaling inhibits melanoma and improves response to immune checkpoint blockage. eLife. 7:e31770.
4. Qiao Liu, Zhuojia Chen, Guanmin Jiang, Yan Zhou, Xiangling Yang, Hongbin Huang, Huanliang Liu, Jun Du, Hongsheng Wang. May 2017. Epigenetic down regulation of G protein-coupled estrogen receptor (GPER) functions as a tumor suppressor in colorectal cancer. Molecular Cancer. 16(1):87.
5. Guangfa Zhu, Yan Huang, Chunting Wu, Dong Wei, Yingxin Shi. August 2016. Activation of G-protein-coupled estrogen receptor inhibits migration of human non-small cell lung cancer cells via IKK-β/NF-κB signals. DNA Cell Biology. 35:343-442.
6. Patrick J Medina and Val R. Adams. January 2016. PD-1 Pathway Inhibitors: immuno-oncology agents for restoring antitumor immune responses. Pharmacotherapy. 36(3):317-334.
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