Glyphosate Alters Mammary Lobular and Ductal Epithelial Cell Differentiation and Triggers Tumor Initiation Events

Abstract

Cancer remains a threat for patients around the world, with an estimated 19.3 million peo-ple diagnosed in 2020. Specifically, breast cancer has been recently found to be the most prominently diagnosed cancer globally, accounting for 15.5% of deaths related to cancer in females. The cellular change toward oncogenesis is closely linked to environmental fac-tors such as herbicides, tobacco, and alcohol. Glyphosate is the most widely used herbicide globally, with usage increasing over the years, from 16 million kg spread in the world in 1994 to 79 million kg spread in 2014, including 15% in the United States alone. The International Agency for Research on Can-cer (IARC) classified glyphosate as "probably carcinogenic to humans" (Group 2A) in March 2015, based on "limited" evidence of cancer in humans and "sufficient" evidence of cancer in experimental animals. Although some studies have looked into its effect on the progression of cancer in tumor-igenic cells, it is still unknown whether glyphosate may initiate cancer and disrupt differ-entiation in normal cells. In this study, we opted to investigate the impact of glyphosate in initiating tumor-like phenotypes in non-tumorigenic estrogen-positive lobular mouse (SCp2) and ductular human estrogen-negative (HMT-3522 S1) breast epithelial cells and disrupting differentiation markers. Our results show that long-term glyphosate exposure did not affect growth rate, but enhanced the invasion compared to the untreated control (~8-fold increase upon 10-11 M glyphosate; p <0.05), matrix metalloproteinase-9 (MMP-9) release, and triggered a 4-fold miR-183 upregulation (preliminary), the most overex-pressed miRNA in early-stage Lebanese breast cancer patients, in SCp2 cells. Long-term as well as short-term glyphosate treatments of human derived S1 cells exhibited an en-hanced cell invasion across a reconstituted basement membrane, whereas long-term expo-sure disrupted lumen formation in their 3D cultures, compared to non-treated cells (~1.3-fold and 1.2-fold increase upon 10-5 and 10-11 M glyphosate, respectively; p <0.05). Short and long-term treatments with glyphosate disrupted the distribution of cell polarity marker β-catenin from and apical to a basolateral distribution. Moreover, we showed the effect of the herbicide on the differentiation markers of SCp2 and S1 cells by evaluating levels of β-casein expression and assessing the effect of treatment on lumen formation, respectively. β-casein expression significantly decreased upon 10-11 M glyphosate treat-ment (p <0.05). We additionally propose a possible pathway for the effect of glyphosate on the activation of breast cancer-related signaling pathways using Ingenuity Pathway Analysis (IPA) in estrogen-dependent cells. In conclusion, our findings may provide insight on the function of glyphosate in tumor initiation events, suggesting that such chemicals might "injure" nontumorigenic breast epi-thelial cells. Further research may be necessary to fully understand the extent of these ef-fects and to develop alternative methods for weed control that do not rely on glyphosate.