Introduction

A broad range of biological functions including the sleep/wake cycle, the endocrine system, and metabolism, exhibit circadian rhythms that are precisely controlled by molecular circadian clocks. Chronic circadian misalignment leads to the development of a wide range of diseases including cancer. Strikingly, a higher risk of many types of cancer is found in shift workers. Determining the interplay between circadian misalignment and tumorigenesis is of high significance to preventing shift workers from developing cancer and establishing novel therapeutic strategies for the treatment of cancer. The circadian clock links to tumorigenesis via regulating cell signaling, cell cycle, and metabolic reprogramming, although the role of the circadian clock in cancer stem cells (CSCs) has not been fully elucidated. Several works have elucidated the crucial roles of the circadian clock Cryptochrome1 (CRY1) in tumorigenesis in vivo and in vitro, all of which studied the functions of CRY1 in bulk cancer cells. Given our previous study found that CRY1 regulates stemness in pluripotent stem cells, we hypothesized that CRY1 plays distinct roles in CSCs vs. differentiated cancer cells (DCCs).

Methods

Aim 1: Uncovering the role of CRY1 in the regulation of breast CSC and DCC heterogeneity.

Breast cancer (BC) is a collection of diseases with complex inter-and intra-tumoral heterogeneities. BC subtypes are a reflection of the different cells of origin, whereas intra-tumoral heterogeneity refers to variation within the tumor due to CSCs, resulting in differing functional properties and markers that may account for relapse and recurrence. We aim to determine whether CRY1 regulates breast CSC stemness, differentiation potency, metabolism, and breast CSC and DCC heterogeneity. BC cell lines, including SUM159 human BC cells and 4T1 mouse BC cells, underwent pharmacological CRY1 activation followed by FACS analysis to isolate CSC and DCC subpopulations. We will also intend to introduce a genetic Cry1 deletion. Breast CSCs and DCCs undergoing genetic and pharmacological intervention targeting CRY1 will be processed for RNA-seq and metabolomics analysis.

Aim 2: Determining whether CRY1 intervention in breast CSCs influences longevity, BC heterogeneity, tumorigenesis, metastasis, and metabolism.

We aim to determine in vivo effects of CRY1 in tumor development and spontaneous metastasis originating from breast CSCs. We will use mouse breast CSCs isolated from 4T1 mouse BC cells lacking Cry1 for orthotopic implantation into female BALB/c mice. The mice will be sacrificed 10 weeks post-implantation, and tumors, along with several organs such as lung and liver, will be harvested to evaluate tumor development and metastasis. To examine the role of CRY1 in tumor development and BC heterogeneity, isolated tumors will be quantified and further processed for scRNA-seq at TAMU single-cell analysis core. Harvested organs will be used for RNA extraction to quantify the levels of tumor biomarkers to evaluate metastasis.

Results

CRY1 activator KL001 significantly inhibited the growth of SUM159 cells, indicating that CRY1 may indeed be a promising target for BC development and therapy. Furthermore, KL001 changed the proportion of CSC and DCC subpopulations in SUM159 cells. Notably, the CSC subpopulation was decreased while the DCC subpopulation was increased in response to KL001 treatment. These results suggest that CRY1 might be involved in regulating stem cell differentiation and self-renewal in BC. Specifically, the decrease in the CSC subpopulation in response to KL001 treatment underscores the possible role of CRY1 in suppressing metastasis and subsequent recurrence. We expect to obtain additional results by October 2023.

Conclusion

This study will enable us to assess whether CRY1 potentially serves as a target molecule for BC therapy via modulating CSC signatures. Together, this study has the potential to contribute to the advancement of BC treatment and prevention.