Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells.Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted.Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model.Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Background Protoporphyrin IX (PpIX) gets accumulated preferentially in 5-aminolevulinic acid (5-ALA)-treated cancer cells. Photodynamic therapy (PDT) utilises the accumulated PpIX to trigger cell death by light-induced generation of reactive oxygen species (ROS). We previously demonstrated that oncogenic Ras/MEK decreases PpIX accumulation in cancer cells. Here, we investigated whether combined therapy with a MEK inhibitor would improve 5-ALA-PDT efficacy. Methods Cancer cells and mice models of cancer were treated with 5-ALA-PDT, MEK inhibitor or both MEK inhibitor and 5-ALA-PDT, and treatment efficacies were evaluated. Results Ras/MEK negatively regulates the cellular sensitivity to 5-ALA-PDT as cancer cells pre-treated with a MEK inhibitor were killed more efficiently by 5-ALA-PDT. MEK inhibition promoted 5-ALA-PDT-induced ROS generation and programmed cell death. Furthermore, the combination of 5-ALA-PDT and a systemic MEK inhibitor significantly suppressed tumour growth compared with either monotherapy in mouse models of cancer. Remarkably, 44% of mice bearing human colon tumours showed a complete response with the combined treatment. Conclusion We demonstrate a novel strategy to promote 5-ALA-PDT efficacy by targeting a cell signalling pathway regulating its sensitivity. This preclinical study provides a strong basis for utilising MEK inhibitors, which are approved for treating cancers, to enhance 5-ALA-PDT efficacy in the clinic.

The efficacy of aminolevulinic acid (5-ALA)-based photodynamic diagnosis (5-ALA-PDD) and photodynamic therapy (5-ALA-PDT) is dependent on 5-ALA-induced cancer-specific accumulation of protoporphyrin IX (PpIX). We previously reported that inhibition of oncogenic Ras/MEK increases PpIX accumulation in cancer cells by reducing PpIX efflux through ATP-binding cassette sub-family B member 1 (ABCB1) and ferrochelatase (FECH)-catalysed PpIX conversion to haem. Here, we sought to identify the downstream pathways of Ras/MEK involved in the regulation of PpIX accumulation via ABCB1 and FECH. First, we demonstrated that Ras/MEK activation reduced PpIX accumulation in RasV12-transformed NIH3T3 cells and HRAS transgenic mice. Knockdown of p90 ribosomal S6 kinases (RSK) 2, 3, or 4 increased PpIX accumulation in RasV12-transformed NIH3T3 cells. Further, treatment with an RSK inhibitor reduced ABCB1 expression and increased PpIX accumulation. Moreover, HIF-1α expression was reduced when RasV12-transformed NIH3T3 cells were treated with a MEK inhibitor, demonstrating that HIF-1α is a downstream element of MEK. HIF-1α inhibition decreased FECH activity and increased PpIX accumulation. Finally, we demonstrated the involvement of RSKs and HIF-1α in the regulation of PpIX accumulation in human cancer cell lines. These results demonstrate that the RSK-ABCB1 and HIF-1α-FECH axes are the downstream pathways of Ras/MEK involved in the regulation of PpIX accumulation.

1The efficacy of aminolevulinic acid (5-ALA)-based photodynamic diagnosis (5-ALA-PDD) and 2 photodynamic therapy (5-ALA-PDT) is dependent on the 5-ALA-induced cancer-specific 3 accumulation of protoporphyrin IX (PpIX). We previously reported that inhibition of oncogenic 4 Ras/MEK increases PpIX accumulation in cancer cells by reducing PpIX efflux through ATP-binding 5 cassette sub-family B member 1 (ABCB1) as well as PpIX conversion to heme by ferrochelatase 6 (FECH). Here, we sought to identify the downstream pathways of Ras/MEK involved in the 7 regulation of PpIX accumulation via ABCB1 and FECH. First, we demonstrated that Ras/MEK 8 activation reduced PpIX accumulation in RasV12-transformed NIH3T3 cells and HRAS transgenic 9 mice. Knockdown of p90 ribosomal S6 kinases (RSK) 2, 3, or 4 increased PpIX accumulation in the 10 RasV12-transformed NIH3T3 cells. Further, treatment with an RSK inhibitor reduced ABCB1 11 expression and increased PpIX accumulation. Moreover, HIF-1α expression was reduced when the 12 RasV12-transformed NIH3T3 cells were treated with a MEK inhibitor, demonstrating that HIF-1α is a 13 downstream element of MEK. HIF-1α inhibition decreased the activity of FECH and increased PpIX 14 accumulation. Finally, we demonstrated the involvement of RSKs and HIF-1α in the regulation of 15 PpIX accumulation in human cancer cell lines (DLD-1, SNB-75, Hs 578T, and MDA MB 231). These 16 results demonstrate that the RSK-ABCB1 and HIF-1α-FECH axes are the downstream pathways of 17 Ras/MEK involved in the regulation of PpIX accumulation. 18 19