# Mechanisms of tumor cell clustering in breast cancer metastasis

> **NIH NIH R01** · BAYLOR COLLEGE OF MEDICINE · 2024 · $439,719

## Abstract

Metastasis is the primary cause of breast cancer-related morbidity and mortality. During metastasis, cells from
primary tumors shed into the bloodstream as circulating tumor cells (CTCs). CTCs travel to distant organs to
establish secondary tumors. CTCs that form clusters exhibit a drastic increase in metastatic potential compared
to single CTCs. While previous studies have described E-cadherin as a key mediator of adhesion in CTC clusters
and metastasis, many breast tumors, including tumors of the highly metastatic triple negative breast cancer
(TNBC), express little to no E-cadherin. Thus, an alternative, E-cadherin-independent mechanism must exist to
mediate clustering between CTCs, thereby promoting metastasis and disease progression. To investigate CTC
clustering mechanisms, we developed an in vitro tumor cell clustering assay that incorporates physiological
shear force and mimics in vivo conditions. Using this assay, we found that E-cadherin-negative metastatic breast
cancer cells can form cellular interactions with characteristics similar to cell adherens junctions. Speculating that
extracellular matrix (ECM) components from tumor cells may contribute to CTC clustering, we analyzed ECM
components by breast cancer subtypes. We found that hyaluronic acid synthase 2 (HAS2), which is the primary
enzyme responsible for hyaluronic acid (HA) production in breast cancer cells, is significantly upregulated in
TNBC. We further observed that HA mediates clustering between TNBC tumor cells and confers them with the
ability to overcome insults present in the bloodstream, including shear forces. Importantly, we detected HA
enrichment at the cell-cell junction of interacting CTCs in TNBC patient blood specimens. Mechanistically, our
preliminary results suggest that metastatic TNBC cells upregulate HAS2 expression in a chromatin looping
mechanism mediated by PRDM6, a transcriptional regulator and putative histone lysine methyltransferase.
Collectively, these preliminary findings lead us to hypothesize that in aggressive TNBC, high levels of HA
augment CTC clustering via HA-dependent adhesive interactions between neighboring cells. We further
hypothesize that the PRDM6 upregulates HA levels through epigenetic modulation of HAS2 expression,
including chromatin looping interactions. We propose to study our hypotheses through two specific aims: 1)
determine the role of HA in TNBC tumor cell clustering and metastasis and 2) elucidate the epigenetic regulation
of HAS2 that impacts breast tumor cell clustering. To investigate our hypotheses, we will utilize novel in vitro
clustering assays and in vivo mouse models along with electron microscopy to reveal the structural
characteristics of CTC clusters. In addition, we will utilize integrative multi-omics data analysis to elucidate the
co-regulatory network governing HAS2 expression during CTC clustering. Importantly, our results will be
extensively validated in blood specimens from metastatic TNBC patients. Conse...

## Key facts

- **NIH application ID:** 10895494
- **Project number:** 5R01CA276432-02
- **Recipient organization:** BAYLOR COLLEGE OF MEDICINE
- **Principal Investigator:** Chonghui Cheng
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $439,719
- **Award type:** 5
- **Project period:** 2023-07-28 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10895494

## Citation

> US National Institutes of Health, RePORTER application 10895494, Mechanisms of tumor cell clustering in breast cancer metastasis (5R01CA276432-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10895494. Licensed CC0.

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