New evidence about the role of the RSK family of protein kinases in cancer has cut through conflicting experimental data to demonstrate they have different functions and that the RSK4 isoform is a promoter of drug resistance and metastasis in lung and bladder cancer.

Silencing RSK4, either through RNA interference or CRISPR gene editing, sensitized tumor cells to chemotherapy and interfered with metastatic processes in vitro and in vivo.

Subsequent drug screening revealed floxacin antibiotics are potent RSK4 inhibitors and they reproduce the effects of RSK4 silencing. A fresh analysis of historical evidence from a large placebo-controlled phase III trial, in which levofloxacin was assessed to see if it prevented or controlled early infection in patients receiving chemotherapy for solid tumors, showed those receiving the antibiotic had significantly increased survival (P = 0.048).

"We are currently seeking funding to initiate a clinical trial combining levofloxacin with chemotherapy in lung adenocarcinoma patients stratified for RSK4 expression, as a proof of principle that inhibiting RSK4 is beneficial in the clinic," said Olivier Pardo of the department of surgery and cancer at Imperial College London, who is group leader on the research, published in the July 14 edition of Science Translational Medicine.

"At the same time, we are pursuing research on novel RSK4 inhibitors derived from floxacin that we hope will be more potent and devoid of antibiotic activity," Pardo told BioWorld Science.

As the paper notes, as a result of the early development of drug resistance and metastasis, lung and bladder cancers are "mostly incurable." Pardo's work in elucidating the mechanisms that underlie this has homed in on the role played by four p90 ribosomal protein S6 kinases (RPS6KA; also referred to as RSK) in these processes.

RSK1 and RSK2 have been most studied, and based on a high degree of gene sequence similarities, it was assumed the biological functions of the other members of the family, RSK3 and RSK4, would overlap.

However, earlier investigations by Pardo and colleagues had begun to challenge this, by showing silencing of RSK1 promoted metastases in vitro and also that RSK4 differs from the rest of the family in its activation mechanism. That led the researchers to hypothesize RSK4 has different functions from other isoforms.

Previous research is equivocal, with some studies suggesting RSK4 has a tumor suppressor function, while other work points to a tumor promoter effect.

Pardo's latest research shows lung and bladder cancer cells in which RSK4 is silenced are sensitive to taxol and cisplatin chemotherapy. The silenced cells did not exhibit any decrease in viability in the absence of these drugs.

Conversely, inducing RSK4 overexpression made the cells resistant to chemotherapy.

In multiple non-small cell lung cancer (NSCLC) cell lines and in bladder cancer cell lines, this sensitization to chemotherapy was associated with decreased expression of antiapoptosis proteins. These proteins previously have been shown to promote drug resistance in lung cancer.

The researchers found also that RSK4 knockdown hindered the migration and invasion of bladder and lung cancer cell lines, whereas overexpression of RSK4 promoted cell migration.

Decreased migration after RSK4 silencing was rescued by RSK4 overexpression. Taken together the cell line data imply RSK4 silencing impairs the ability of cells to migrate and metastasize.

To assess potential clinical relevance, the researchers next analyzed normal and lung cancer tissue, finding while RSK4 is undetectable in normal lung, 57% of 97 primary lung cancer specimens were positive for the kinase.

Bolstering this, publicly available lung cancer survival data indicate higher RSK4 expression is associated with poorer prognosis. The association between RSK4 expression and overall survival was noted despite the fact that RSK4 expression was far lower than RSK1, RSK2 and RSK3 expression across all lung cancers. Further strengthening the case for targeting RSK4, a negative association between RSK expression and survival was only observed for RSK4.

On the other hand, high expression of RSK1 did not affect overall survival, while high expression of RSK2 and RSK3 was associated with higher overall survival. Similar results were found in bladder cancer.

"RSK1 ... shows opposite biological functions [to] those of RSK4. Indeed, inhibiting RSK1 promotes drug resistance and metastasis," Pardo said. "RSK2 and RSK3, the two last members of the family, had less consistent biological activity, with RSK2 inhibition sometimes impacting invasiveness similarly to RSK4, and RSK3 being essentially inactive in our assays."

The net result of inhibiting RSK1 and RSK4 together "either averages out the biological output of both kinases, with no benefit, or reproduces the effects of inhibiting RSK1 singly, which would be unwelcome in a therapeutic setting," said Pardo. "Hence, it is imperative to specifically target RSK4 to obtain therapeutic benefit, something that floxacins appear to provide."

In mouse models, RSK4 silencing impaired tumor progression and increased the response to chemotherapy.

Although there are pan-RSK inhibitors, in advance of this research project there were no known RSK4-specific inhibitors.

Small-compound screening led the researchers to floxacins and then to trovafloxacin as best reproducing the effects of RSK4 silencing in vitro. Further studies uncovered the specifics of the binding site on RSK4, paving the way for de novo drug discovery.

"We have already synthesized derivatives of trovafloxacin that retain RSK4 inhibitory activity and will soon start testing them in a variety of assays to assess whether we have successfully weeded out antibiotic activity and improved on RSK4 inhibitory potency in cells," said Pardo. "It is likely that several more cycles of medicinal chemistry will be needed to obtain compounds that are ready for clinical testing."

In advance of any clinical research of their own, the researchers were able to show the related antibiotic levofloxacin prolongs survival in solid tumors in a retrospective analysis of a 1,500-patient trial conducted in Birmingham, U.K., to determine if prophylactic treatment with the antibiotic reduces the rate of clinical infection in patients receiving myelosuppressive chemotherapy (ClinicalTrials.gov Identifier NCT00005590).

Overall survival was significantly longer in the levofloxacin arm, at 72 months, versus 59 months in the placebo arm. The trial included patients with 8 different types of solid tumors. The hazard ratio was better for patients with lung and bladder cancer compared with the entire cohort.

"Beyond lung and bladder cancers, we did not yet have the opportunity to look at each tumor type," Pardo said. For some, the number of patients involved might be too small to provide statistical significance. "Nevertheless, our preliminary in vitro data suggest that RSK4 is also a tumor promoter in additional cancer types" said Pardo.

"Reanalysis of the data from the trial, while encouraging, must be taken cautiously, as inhibition of RSK4 may not be the only factor resulting in improved patient survival. This is why we are now looking for funding to initiate a dedicated clinical trial to stringently assess how RSK4 inhibition impacts on treatment response and overall survival of patients with lung cancer," Pardo said.

The researchers also intend to continue their basic research into the biological roles of RSK4 in cancer and beyond. "We especially want to reveal the molecular mechanisms responsible for the opposing biological functions of RSK1 and RSK4, and elucidate the role of RSK4 variants in different cancers," said Pardo.