1) Breast cancer stem cells (BCSCs) are responsible for tumor development and maintenance due to their self-renewal and survival abilities, and they have high resistance to treatments like chemotherapy. 2) The study found that signaling between the Ron receptor tyrosine kinase and its ligand HGFL enhances the therapeutic resistance of BCSCs by increasing their proliferation, survival, and ability to form mammospheres (clusters of stem cells). 3) Inhibiting Ron signaling, such as with a Ron inhibitor combined with chemotherapy, reduced BCSC proliferation and mammosphere formation, suggesting it may help eradicate treatment-resistant BCSCs and improve patient outcomes.

1. HGFL-Ron Signaling Enhances the Therapeutic Resistance of Breast Cancer Stem Cells
Abstract
Breast cancer stem cells (BCSCs) are a subpopulation of cancer cells that
are responsible for the development and maintenance of breast cancer
due to their specific capabilities to self-renew, proliferate, and survive.
BCSCs also have high resistance to standard treatments, such as
chemotherapy and radiotherapy, leading to relapse. Reports demonstrate
that the Ron receptor tyrosine kinase and its sole ligand, the Hepatocyte
Growth Factor-Like (HGFL) protein, promote breast cancer by increasing
epithelial breast cancer cell growth, motility, and survival. In extending
our understanding of how Ron signaling promotes breast cancer,
preliminary studies suggest that HGFL-Ron signaling enhances BCSC
populations, in part by increasing BCSC numbers and their self-renewal
ability. However, the role of the HGFL-Ron signaling in regulating the
resistance of BCSCs to different therapies has not been investigated. To
test the function of HGFL-Ron signaling in regulating BCSC therapeutic
resistance, Ron deficient and proficient BCSCs were treated with different
chemotherapeutic agents and their ability to form mammospheres was
evaluated. Data demonstrate that HGFL-Ron signaling enhances BCSC
resistance to the tested chemotherapies. Next, we evaluated the cellular
mechanisms induced by Ron signaling to promote BCSC therapeutic
resistance. Flow cytometry analyses for proliferation and survival markers
suggest that HGFL-Ron signaling promotes BCSC therapeutic resistance by
increasing their proliferation and survival. Overall, our data provide (i) the
first evidence demonstrating both HGFL and Ron as factors regulating
BCSC therapeutic resistance and (ii) that this occurs by increasing BCSC
proliferation and survival. These results suggest that use of combination
therapies, such as Ron inhibitors and chemotherapies, may aid in
eradicating BCSCs and improve the outcome of breast cancer patients.
Methods
•Mouse breast cancer cell lines: R7 (express Ron and HGFL), R7shRon, and R7shHGFL
•Western Blot and quantitative real-time PCR (qRT-PCR) characterized Ron and HGFL expression
•Cell viability assays determined the chemotherapy concentrations to be used in mammosphere
(MS) formation assays
•BCSC therapeutic resistance was tested through BCSC-enriched mammosphere formation
assays
•BCSC survival and proliferation were assessed by flow cytometry analyses (AnnexinV/PI, BrdU)
Holly E. Kraus1,2
, Sasha J. Ruiz-Torres1
, and Susan E. Waltz, PhD1,3
1
Department of Cancer Biology, University of Cincinnati College of Medicine, 2
Wheeling Jesuit University, and 3
Research Service, Cincinnati Veterans Hospital Medical Center,
Cincinnati, OH 45267
Results
Figure 4. HGFL-Ron signaling enhances BCSC proliferation and BMS-777607 treatment makes the Ron signaling
BCSCs act similar to Ron deficient BCSCs. Percent of proliferating cells (BrdU+) after 10uM Cisplatin (A), 10uM
Doxorubicin (B), and 100uM BMS-777607 (C) treatment. *P<0.05
A B C
Figure 2. HGFL-Ron signaling in BCSCs promotes mammosphere formation and resistance after
chemotherapy treatment. A) Representative picture of R7 mammospheres. B-D) Quantification of the
number of mammospheres formed after 10uM Cisplatin (B), 10uM Doxorubicin (C), and 100uM BMS-
777607 (D) treatments. *P<0.05
A B
Hypothesis
HGFL-Ron signaling enhances the therapeutic resistance of breast cancer stem cells by
increasing their mammosphere formation, proliferation, and survival capabilities
Conclusions
•Ron signaling enhances proliferation, inhibits apoptosis, and promotes mammosphere formation of
BCSCs
•BCSCs with genetic ablation of Ron signaling exhibit reduced proliferation, survival, and
mammosphere formation abilities; therapies targeting Ron reduce BCSC mammosphere formation
and
proliferation
•BCSC therapeutic resistance was more affected by a genetic loss of Ron signaling and was
marginally affected by chemotherapy
•Our studies support targeting Ron to reduce the number and properties of BCSCs
Future Work
•Reproduce mammosphere formation assay experiments
•Examine the sensitivity of Ron expressing BCSCs to other traditional chemotherapies
•Determine the mechanisms by which Ron promotes therapeutic resistance in BCSCs – STAT1, β-
catenin
•Assess the response of Ron-expressing BCSCs to chemotherapy following orthotopic transplantation
into mice
Acknowledgements
I would like to thank the University of Cincinnati, the ASPET-SURF Dalton Zannoni summer fellowship
A
A
C D
C
B
Figure 3. HGFL-Ron signaling promotes BCSC resistance to Cisplatin and Doxorubicin by inhibiting cell apoptosis.
Percent of alive, early apoptotic, late apoptotic, and dead cells obtained based on the expression of Annexin
V/Propidium Iodide markers after 10uM Cisplatin (A) and 10uM Doxorubicin (B) treatment.
Introduction
Breast Cancer Stem Cells (BCSCs)
•Tumor initiating cells, have high self-renewal,
proliferation, and survival abilities - essential
for tumor initiation/development
•Have enhanced resistance to treatments,
problematic to target in the clinic
* *
D
HGFL-Ron Signaling
•Ron – receptor tyrosine kinase activated by its
ligand, HGFL
-Upregulated in several cancers, including breast
cancer
-Enhances tumor cell growth, survival, motility,
and angiogenesis
Chemotherapeutic Agents
•Pro-apoptotic: Cisplatin, Doxorubicin
•Anti-proliferative: BMS-777607
B
Results
Figure 1. Ron and HGFL expression increase in R7 BCSC-enriched mammospheres compared to
parental R7 cells, while R7shRon and R7shHGFL cells are deficient in HGFL-Ron signaling. qRT-PCR
analyses show increased Ron (A) and HGFL (B) expression in R7 BCSC-enriched mammospheres (R7
MS). (C) Western analyses for Ron in cell lines with alterations in Ron expression. (D) qRT-PCR analysis
showing lower HGFL expression in R7shHGFL cell line.