Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling

Abstract Metabolic reprogramming plays important roles in development and progression of nasopharyngeal carcinoma (NPC), but the underlying mechanism has not been completely defined. In this work, we found INSL5 was elevated in NPC tumor tissue and the plasma of NPC patients. Plasma INSL5 could serve as a novel diagnostic marker for NPC, especially for serum VCA‐IgA‐negative patients. Moreover, higher plasma INSL5 level was associated with poor disease outcome. Functionally, INSL5 overexpression increased, whereas knockdown of its receptor GPCR142 or inhibition of INSL5 reduced cell proliferation, colony formation, and cell invasion in vitro and tumorigenicity in vivo. Mechanistically, INSL5 enhanced phosphorylation and nuclear translocation of STAT5 and promoted glycolytic gene expression, leading to induced glycolysis in cancer cells. Pharmaceutical inhibition of glycolysis by 2‐DG or blockade of INSL5 by a neutralizing antibody reversed INSL5‐induced proliferation and invasion, indicating that INSL5 can be a potential therapeutic target in NPC. In conclusion, INSL5 enhances NPC progression by regulating cancer cell metabolic reprogramming and is a potential diagnostic and prognostic marker as well as a therapeutic target for NPC.

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Please click the link below to provide an ORCID: Link Not Available 12) The system will prompt you to fill in your funding and payment information. This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance. This quote takes into account any reduction or fee waivers that you may be eligible for. Authors do not need to pay any fees before their manuscript is accepted and transferred to our publisher. Needs additional experiments as detailed in the report Referee #1 (Remarks for Author): The manuscript by Li and colleagues is an interesting piece of work convincingly showing the potential of the insulin-like peptide 5 (INSL5) as biomarker of diagnosis and of bad prognosis of patients bearing nasophayngeal carcinomas (NPC). Less developed are the aspects related with the mechanisms that underlie the pro-oncogenic role of INSL5. In this regard, they overexpressed INSL5 or silenced its high-affinity receptor, the G-protein coupled receptor RXFP4 (GPCR142), in different cellular lines to demonstrate that INSL5, or its downstream signaling, favor in vitro proliferation and invasion of the cells and in vivo tumor growth in xenograft mouse models. In the search for a mechanism of action of INSL5, they report that both mRNAs, enzymes and metabolites of the glycolytic pathway are significantly increased upon the action or signaling through INSL5, whereas oxidative phosphorylation and metabolites of the TCA cycle negatively correlate with the activity of INSL5. These and some other additional results led the authors to suggest that INSL5 mechanism of action is through transcriptional upregulation of glycolysis. Mechanistically, they report that INSL5 triggers the phosphorylation and nuclear translocation of transcription factor STAT5, which seems to interact with the promotes of some glycolytic genes and increase their activity upon INSL5 stimulation. Finally, they show that antibodies against INSL5 or its receptor GPCR142 diminish the pro-oncogenic activity of INSL5 in patient-derived tumor xenografts suggesting that they could provide targets for the treatment of NPC. Overall, the paper needs of additional experiments to convey a convincing message. Major points 1. INSL5-and RXFP4-mediated metabolic reprogramming are poorly characterized and their effects on mitochondrial respiration and oxidative phosphorylation need to be reported. Is the cellular mitochondrial content affected? 2. The two-fold increase in cellular ATP content in response to the overexpression of INSL5 (Fig. 4I) or its sharp decrease in knock-down cells (Fig. 4K) is difficult to reconcile with the marginal changes published in cellular ATP content. What is the effect of 2DG and of oligomycin in cellular ATP content? 3. The phosphorylation and nuclear translocation experiments of STAT5 in response to INSL5 (Fig.  5B-D) are not convincing. They require quantification and the incorporation of the results obtained in histograms in the same figure. STAT5 is known to activate anti-apoptotic genes. Is any of these anti-apoptotic genes activated in response to INSL5? What is the contribution of preventing cell death in tumor accretion mediated by INSL5 action? 4. The INSL5/RXFP4 downstream signaling cascade to STAT5 phosphorylation needs to be delineated. Which kinase (ERK, JAK, Akt...) is involved? 5. Apparently, blocking glycolysis with 2DG has no relevant effect in tumor growth (Fig. 6F), raising doubts about the relevance of glycolytic reprogramming in the INSL5-mediated pro-oncogenic role. The authors should consider combining the treatment of blocking INSL5 or GPCR142 antibodies with conventional chemotherapy (cisplatin, carboplatin, 5FU or docetaxel) regimens of NPC. 6. Is the INLS5 knock-out mice resistant to the growth of NPC?
Minor points There are some spelling errors that need amendment in the text (line 131, OASL mRNA....) and in the figures (Fig. 4K, reads Akt when it should be ATP) Explain why the 3.73 cut-off value has been selected (line 185) in KM analyses The mouse genes need to be appropriately quoted.

Referee #2 (Comments on Novelty/Model System for Author):
Under my knowledge, INSL5 has not been before involved in the progression of NPC. Moreover, it is shown in this study that INSL5 can be a prognosis and diagnosis marker of this cancer type. This opens the door also to analyze this protein other cancers.

Referee #2 (Remarks for Author):
Li et al. describe in this paper a new role for INSL5 in the progression of nasopharyngeal carcinoma. They start by proving the use of INSL5 quantification as a diagnostic and prognostic marker of NPC in human patients. Next, they use cellular models of NPC to analyze the underlying mechanisms by which INSL5 regulates the proliferation and growth of these cancer cells. They show, using different approaches, that INSL5 through binding to its receptor GPCR142, activates the STAT5 pathway to regulate the expression of genes involved in the control of glycolysis. They claim that through this process, INSL5 facilitates the metabolic switch required for cancer cells to proliferate. The study is very extensive, and covers from studies in human patients of cancer to work in mouse models. Furthermore, through the use of cellular models they unravel the molecular mechanisms underlying the effects of INSL5 on cancer cells. This is a novel and original work that has been well conducted.
There is no doubt about the relevance of this study for cancer research and treatment. Some additional work, as is next suggested, would further improve the quality of the manuscript and reinforce the conclusions. 4. Several reports suggest that there is an important correlation between the JAK/STAT pathway and cell metabolism, notably the activation of hypoxia-inducible factors and the alteration of mitochondrial activity. In addition to the regulation of glycolysis genes, STAT5 has also been involved in mitochondrial function. A more detailed functional evaluation of mitochondria and glycolytic function of the cells with or without INSL5 (using for instance Seahorse analysis) would further prove the direct involvement of this protein in the control of metabolism in these cancer cells.
5. In the Figure 6 A-B, the authors show the effects of INSL5 inhibition by using a monoclonal antibody. Cell death and apoptosis should be analyzed in these cells.

Point-by-point response
We thank all the reviewers for their constructive comments and suggestions. We have performed additional experiments and revised the manuscript accordingly. We believe that we have addressed all the concerns raised by reviewers in the revised manuscript.
All changes in the revised manuscript are highlighted in yellow for your attention.
The following is a point-by-point response on how we revised our manuscript. Mechanistically, they report that INSL5 triggers the phosphorylation and nuclear translocation of transcription factor STAT5, which seems to interact with the promotes of some glycolytic genes and increase their activity upon INSL5 stimulation. Finally,  Fig S5G) and impaired oxidative phosphorylation (OXPHOS) (Fig S5H), which indicated that INSL5 overexpression could promote metabolism shift from OXPHOS to aerobic glycolysis.
2. The two-fold increase in cellular ATP content in response to the overexpression of INSL5 (Fig. 4I) or its sharp decrease in knock-down cells (Fig. 4K) (Fig 4J and Fig S5K). Thanks for your helpful suggestion. For Fig.5B-D, we quantified the western blotting results by Image J and labeled the fold change just above the indicated bands. We agreed with you that STAT5 is known to activate anti-apoptotic genes, like c-myc, BCL2 and BCL-xL. We detected those genes in INSL5 overexpressing and control cells, and found that INSL5 overexpression only increased c-myc expression, not BCL2 and BCL-xL (Fig S6D). We also detected cell apoptosis under conventional chemotherapy (5-FU and DDP), the results showed that INSL5 overexpression suppressed the sensitivity of NPC cells to 5-FU or DDP. Furthermore, we detected the apoptosis pathway, and found that INSL5 overexpression could suppress the cleavage of caspase 3, caspase7 and caspase 9. Taken together, all of those data suggested that INSL5 overexpression could promote chemoresistance to 5-FU or DDP via inhibiting cell apoptosis (Fig S6E-G). Response: Thanks for your great suggestion. We agree that glycolysis inhibitor 2-DG only shows minor effect in tumor growth. As suggested, we examine whether 2-DG could reverse INSL5 enhanced chemoresistance. We found that 2-DG could sensitize INSL5 highly expressed NPC to chemotherapy (Fig 6F and Fig S7C). Additionally, we combined the treatment of blocking INSL5 or GPCR142 antibodies with conventional chemotherapy (DDP) in tumor-bearing mice, and we found that INSL5 overexpression displayed chemoresistance to DDP treatment, which can be reversed by INSL5 or GPCR142 antibodies treatment (Fig S7E-G Indeed, in the figure 6F, the growth of the tumors is only minimally changed when glycolysis is inhibited by 2-DG. Response: Thank you very much. We agree with you that metabolic switch is not the only mechanism to underlie the effects of INSL5 in cancer progression. As your helpful suggestion, we also found that INSL5 could enhance cell cycle progression and suppress cell apoptosis (Fig S6). 2-DG had minimal effect on tumor growth, but in our revised manuscript we found that 2-DG could reverse the chemoresistance induced by INSL5, which indicated that 2-DG could sensitize INSL5 expressed NPC to chemotherapy (Fig 6F and Fig S7C). Based on those finding, besides to metabolic switch, we corrected our conclusion that INSL5 induced metabolism reprograming at least partly contributed the accelerated proliferation in the discussion 2. The STAT5 pathway has been involved in tumorigenesis in several types of cancer.
Previous studies show that STAT5 can mediate oncogenic signals and regulate cell cycle progression, proliferation and promote cancer cell survival. Are there other STAT5-regulated pathways, in addition to glycolysis, changed in the models used? Response: Thanks for your great suggestion. Indeed, STAT5 was involved in many oncogenic signals as you mentioned. In the revised manuscript we added the new data about cell cycle, which suggested that INSL5 overexpression could promote cell cycle progression. We also detected the key cyclins and p27, and found that INSL5 overexpression could enhance cyclin D and cyclin E expression, and decreased p27 level (Fig S6A-B). All of those suggested that INSL5 could also promote cell cycle progression in addition to glycolysis.
3. Are JAK kinases, the upstream activators of the STAT pathway also involved? Are JAK kinase inhibitors or STAT5 knock-down experiments abrogate the effects of INSL5? Response: We really appreciate your suggestion. We detected several possible upstream activators (Akt, ERK1/2 and JAK) of STAT pathway and found that INSL5 could increase the phosphorylation of Akt, ERK1/2 as reported, also JAK1 and STAT5.
JAK1 inhibitor and STAT5 knock-down could decrease INSL5 induced cell proliferation and glucose uptake (Fig 5H-J). Taken together, those data suggested that Response: Thanks for your helpful suggestion. We performed Seahorse analysis in three different INSL5 overexpressing cell lines (CNE1, CNE2 and HK1) to detect extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using a XF96 Extracellular Flux analyzer. We demonstrated that INSL5 overexpression significantly increased glycolysis (Fig 4F-G and Fig S5G) and impaired oxidative phosphorylation (OXPHOS) (Fig S5H), which indicated that INSL5 overexpression could promote metabolism shift from OXPHOS to aerobic glycolysis.
5. In the Figure 6 A-B, the authors show the effects of INSL5 inhibition by using a monoclonal antibody. Cell death and apoptosis should be analyzed in these cells.

Response:
We performed the same treatment as Figure 6A-B, and then detected the cell death and apoptosis by flow cytometry after annexin V/propidium iodide (PI) staining. The results showed that the monoclonal antibody along had no effects on cell death and apoptosis ( Fig S7A).
25th May 2020 1st Revision -Editorial Decision 25t h May 2020 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from the two referees who were asked to re-assess it. As you will see the referees are now support ive and I am pleased to inform you that we will be able to accept your manuscript pending the following amendment s.  Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
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