A Bacterial Signaling Molecule Lends Tumors Drug Resistance

Breast cancer is the second highest diagnosed cancer in women in the US and, as of 2020, the most commonly diagnosed cancer in women worldwide.¹ In 15–20 percent of these cases, the growth factor receptor ErbB2, encoded by erythroblastic oncogene B, is overexpressed, increasing the tumor aggressiveness and leading to higher risks of relapse and mortality.² The drug trastuzumab targets this protein, however, up to 35 percent of patients do not respond immediately, and 70 percent will develop resistance after one year.³ The mechanism for this failure is unclear.

One factor that could help researchers better understand tumor growth and therapy resistance is the impact of bacteria from the microbiome and those microbes that live within tumors. “It’s an emerging field,” said Melanie Rutkowski, an immunologist at the University of Virginia.

Among these, Pseudomonas aeruginosa is the most common bacteria found in breast tumors, but its role isn’t well understood.⁴ Xiaohong Fang, a chemist studying molecular medicine at the Chinese Academy of Science, and her group helped elucidate the mechanism of action of a P. aeruginosa signaling molecule, N-(3-oxo-dodecanoyl) homoserine lactone (3oc), that induced apoptosis in immune cells.⁵ After this work, Fang, whose group uses tumor models, explained, “[My graduate student] asked whether 3oc also [had] any effect on the tumor cells].” Fang noted that many drug resistance studies overlooked the contribution of intratumoral bacteria.

Motivated by this gap, the team demonstrated in a new study that 3oc produced by P. aeruginosa indeed contributed to trastuzumab resistance in breast cancer cells.⁶ The findings, published in the Proceedings of the National Academy of Science, offer insights into the role of intratumoral bacteria and their role in chemotherapy resistance.

“This opens the door for really addressing some of these resistant mechanisms that are developing in patients, and how, actually, the microbiome could be a host intrinsic driver of resistance to these therapies,” said Rutkowski, who was not involved in the study.

To investigate 3oc’s effect on chemotherapy resistance, the team treated three breast cancer cell lines with a range of 3oc doses for 72 hours: high ErbB2 expression and trastuzumab sensitive, high EbrB2 expression and trastuzumab resistant, and low ErbB2 expression. The treatment increased drug resistance in all three lines, including that of the previously sensitive cells, in a dose-dependent manner. Unlike in immune cells, where 3oc halts proliferation and induces apoptosis, the researchers observed that this molecule did not influence cell viability.

To determine how 3oc affects breast cancer cells, the researchers compared mRNA expression before and after 3oc treatment. When they assessed differentially expressed genes, the team found that 3oc treatment activated genes in the tumor growth factor β (TGF-β) and ErbB2 pathways.

Trastuzumab inhibits cancer cell growth by binding to ErbB2 to block its signaling, shutting down this vital tumor pathway. The team suspected that 3oc could activate the ErbB2 pathway downstream of its receptor or via TGF-β signaling, overcoming inhibition by trastuzumab. They confirmed that 3oc phosphorylated a signal transducer in TGF-β. Treating cells with a TGF-β inhibitor blocked this effect, which also prevented the stimulation of ErbB2 and its downstream signaling.

Finally, the researchers investigated patient tumor samples for the presence of P. aeruginosa and 3oc. They detected P. aeruginosa by quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH) in seven out of 34 tumors. Tumors from patients who did not fully respond to trastuzumab treatment were more likely to contain bacteria. Separately, they detected 3oc in eight out of 24 tumors by liquid chromatography ionization tandem mass spectrometry. These findings provide clinical support that P. aeruginosa is present in at least some patient tumors and produces 3oc that could drive chemotherapy resistance.

Francesca Gazzaniga, a cancer immunologist at Harvard Medical School who studies the role of the gut microbiome on the immune response to cancer but was not involved in the study, said that the present study demonstrates a potential mechanism for trastuzumab resistance and has interesting implications. However, she added that one challenge in studying the role of bacteria in tumors is being able to confirm that bacteria is present and is not just contamination introduced during preparation, like with tissue blocks. “The thing that we need to look forward in the future is when we're doing these studies, to be very paranoid about contamination, and so really, the whole time thinking, ‘what are my negative controls?’”

Fang’s group used multiple negative controls in their qPCR studies, and they observed P. aeruginosa inside of cells using FISH. Gazzaniga said that being able to confirm that the bacteria in a tumor are indeed living intracellularly increases the chances that the finding is real and not due to contamination. “It's true overall that people need to really think about the possibility of microbes impacting cancer immunotherapies, and probably many other drug treatments,” Gazzaniga said.

Fang added that, because TGF-β activation and ErbB2 signaling are observed in other cancers and resistance mechanisms, her team is interested in exploring whether 3oc plays a role in these systems as well. Additionally, she said that they are interested in studying other bacterial signaling molecules that are similar to 3oc but could have distinct functions. “We can explore this new mechanism and its new applications,” Fang said.

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