Today there are more than 85,000 EPA-registered synthetic chemicals, but only 10% have been tested for
carcinogenicity in animal studies. Of those tested, ~200 have shown evidence of causing cancer and/or an
increase in mammary tumors. However, few of the chemicals have been evaluated in human studies, and results
in humans have been inconclusive. Our study will focus on chemical body burden of per- and poly-fluoroalkyl
substances (PFAS), and concomitant risk of developing invasive breast cancer during the menopausal transition.
These “forever chemicals” are pervasive, enduring, and of high public interest, yet studies of their possible
relationship to breast cancer have been limited. We hypothesize that elevated body-burden levels of these
endocrine-disrupting chemicals (EDCs) will increase the risk of developing invasive breast cancer, and that
alterations in DNA methylation (DNAm) and the breast microenvironment are mechanisms that link these
chemical exposures and breast cancer. We will test this hypothesis using a three-pronged approach. In Specific
Aim 1, we will conduct a prospective study of women in the American Cancer Society Cancer Prevention Study-3
(CPS-3) to assess the association between body burden of PFAS during the menopausal transition and
subsequent development of invasive breast cancer. We will measure PFAS levels in plasma samples collected
1 to 7 years before invasive breast cancer diagnosis in 1000 CPS-3 participants and 1000 matched, cancer-free
CPS-3 participants, all between 40 and 57 years of age at blood draw. In Specific Aim 2, studying the same
participants, we will measure DNAm using Infinium MethylationEPIC BeadChips and conduct an epigenome-
wide association study (EWAS) to identify DNAm changes associated with levels of PFAS in the cancer-free
participants. We then will determine the association between the PFAS-associated DNAm changes and risk of
developing breast cancer. This valuable DNAm data also can be used later for other outcomes, including
exposure to other EDCs. In Specific Aim 3, we will determine the direct effects of PFAS on tissue- and molecular-
level states associated with susceptibility to cancer initiation in genomically-well-characterized primary human
breast mammary epithelial cells (HMECs). Identifying these mechanisms in primary breast cells is critically
important, as PFAS mechanisms of action generally differ by tissue. We will use 2-D cultures to determine the
effects of short-term exposures and 3-D cultures to define the effects of protracted chemical exposures on
changes in epithelial lineage consistent with accelerated aging and age-related molecular changes in genome
methylation, lineage-specific transcription, and cytokeratin proteins. Results from this multi-disciplinary approach
will advance our understanding of the effects of PFAS exposures on risk of developing breast cancer during an
important window of susceptibility. Ultimately, we hope results from our proposed project will identify an
integrated biological signature of environmental exposure, deliver mechanistic insights into breast cancer
development from EDCs, and inform future studies for prevention strategies to reduce or mitigate exposures.
