We began the work by developing unique Y-positive and Y-negative murine cancer cell models for in vitro and in vivo studies through the use of both naturally arising bladder cancer cells with LOY and CRISPR-mediated deletion of the entire Y chromosome in Y-positive tumor cells. With these models, we found that Y-positive and Y-negative tumors grew similarly in vitro, however, Y-negative tumors grew more aggressively than Y-positive tumors in normal mice. To determine the reasons for this difference, we repeated the experiment in the same mice used initially which had a normal immune system, and compared them to genetically engineered (i.e. transgenic) mice that had no immune system. Interestingly, the tumors grew similarly in immune-compromised mice suggesting that Y-negative tumors were somehow better at escaping destruction by the immune system than Y-positive tumors. Further experiments with transgenic mice missing various key parts of the immune system coupled with high-dimensional flow cytometric analyses demonstrated that Y-negative tumors grow better by promoting exhaustion of T cells in the tumor microenvironment. T-cells are one of the most important cell types of the immune system in destroying cancer cells. T-cell exhaustion is a state of the T-cell that ranges from altered functionality of the T-cell to complete lack of its effector function.
The translational and clinical relevance of these findings were validated using RNA sequencing and proteomic evaluation of human bladder cancers. Compared with Y-positive tumors, Y-negative tumors exhibited an increased response to anti-PD-1 immune checkpoint blockade therapy in both mice and patients with cancer. Our study also reviewed the relationship between clinical outcomes and LOY in two distinct patient cohorts. The first was on male patients with muscle invasive bladder cancer who had their bladders removed and were part of The Cancer Genome Atlas Program (TCGA) study. While the second examined male patients from the IMvigor210 atezolizumab (anti-PD-L1) clinical trial. Patients with LOY had poorer prognosis when their bladders were removed, yet better overall survival rates when they received immune checkpoint inhibitors. While this would appear at first to be somewhat paradoxic, it is indeed not since the T-cells in the Y-negative tumors are exhausted and anti-PD-L1 can reverse that condition in part.
We believe this study is the first published evidence that LOY in cancer drives tumor biology making a tumor more aggressive and does so by altering T cell function, allowing bladder cancer to elude the immune system and grow more aggressively. This work provides insights into the biology of LOY, and it lays the groundwork for developing future biomarkers to improve cancer immunotherapy. For example, evaluation of Y chromosome gene expression could be used to select bladder cancer patients for immune checkpoint blockade therapy by determining those who might show superior response and better survival outcomes. More speculatively, these models can be used to discover what additional inhibitors can potentially further enhance tumor response when used in combination with immune checkpoint blockade therapy.
These findings could also have implications for women since they are known to experience more aggressive bladder cancer. The Y chromosome possesses a set of related genes, called Y paralog genes, on the X chromosome, and these might play a role in both women and men. In fact, alterations in some of these genes have already been shown to play a role in cancer in women.
Finally, this research highlights the importance of sex and gender in cancer research and the interplay between the chromosomes and the hormonal microenvironment in cancer and other diseases. It is clear attention should be given to these parameters as we develop new cancer treatments and therapies for all our patients.
Written by: Dan Theodorescu, MD, PhD & Richard Suzuki, PhD, Cedar-Sinai Medical Center, Los Angeles, California.
Graphics by: Jared Schafer, Graphic & Motion Design