HIP1 mediates oncogenic transformation and cancer progression through STAT3 signalling
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Abstract
Background
Huntingtin-interacting protein 1 (HIP1) is an adaptor protein involved in transcriptional regulation and receptor-mediated endocytosis. Overexpression of HIP1 transforms cell and is associated with, increasing grades of prostate cancer (CaP) and poor patient outcomes. However, the precise mechanism for the role of HIP1 in prostate cancer progression remains unknown.
Methods
Using a phospho-kinase antibody array we identified changes in signalling associated with HIP1 overexpression PNT1 cells. For validation Western blots were used together with knockdown or inhibitor treatments and phenotypic assays for cellular transformation. The cell line was xenografted to assess tumour growth. Gene expression microarray analysis of the cell line was used to identify perturbations in transcript levels.
Results
Here we demonstrate cellular transformation and phenotypic effects of HIP1 overexpression in a benign prostate epithelial cell line to be dependent on STAT3 signalling. In vivo xenografts confirmed the cellular transformation phenotype. Gene expression analysis revealed serum protein GDF15 to be a marker of prostate cancer tumorigenesis in our model. We present a HIP1-STAT3-GDF15 axis in our pre-clinical model that mediates cellular transformation and tumorigenesis.
Conclusion
Our findings provide a model defining the functional effects of increased HIP1 expression in prostate tumorigenesis and progression. This model implicates increased STAT3 signalling in HIP1-dependent prostate carcinogenesis and identifies GDF15 as a secreted factor supporting this process. The role of HIP1-STAT3-GDF15 signalling may extend to other epithelial cancers shown to overexpress HIP1; such as gliomas, colon and breast cancer where STAT3 is an emerging oncology drug target.
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###Reviewer #3:
General assessment:
In this research article, authors claim that HIP1 plays an important role in promoting the proliferative ability of prostate cancer cells by HIP1-STAT3-GDF15 signaling axis activation. HIP1 overexpression increased STAT3 signaling in response to FGF2 receptor activation and increased GDF15 transcription. The increase in GDF15 protein secretion was dependent on HIP1 and STAT3 expression and was shown to have paracrine growth-promoting effects. Although some of the information is new, the relevance and importance of this information is inconclusive and not supported from the data presented in this article.
Major Comments:
This paper needs a substantial amount of revision, as indicated below.
A. Novelty:
HIP-1 has been extensively studied in cancer including prostate cancer (Rao et al., 2002). Its role …
###Reviewer #3:
General assessment:
In this research article, authors claim that HIP1 plays an important role in promoting the proliferative ability of prostate cancer cells by HIP1-STAT3-GDF15 signaling axis activation. HIP1 overexpression increased STAT3 signaling in response to FGF2 receptor activation and increased GDF15 transcription. The increase in GDF15 protein secretion was dependent on HIP1 and STAT3 expression and was shown to have paracrine growth-promoting effects. Although some of the information is new, the relevance and importance of this information is inconclusive and not supported from the data presented in this article.
Major Comments:
This paper needs a substantial amount of revision, as indicated below.
A. Novelty:
HIP-1 has been extensively studied in cancer including prostate cancer (Rao et al., 2002). Its role in STAT3 signaling has also been demonstrated (Hsu et al, 2015). This study is not very novel.
B. Major comments:
Figures 1A, S1: Changes in p-AMPK1α, and p-Akt are very profound in this array, however, the authors indicate that "By contrast to our validation of STAT3 phosphorylation by Western blotting, it was not possible to detect increased levels of p-AMPK1α (T174), p-Akt (S473) or p-PLC-γ1 when we attempted to validate these by blotting (Supplementary Figure S1D-F)." Why do the authors think this is happening? Did the authors use the same experimental conditions for the array and validation experiments? These apparent discrepancies need further clarification.
Figure 1E: the authors show that shHIP1#2 caused a modest knockdown of HIP1, while shHIP1#1 induced a dramatic reduction in HIP1 protein level, however, both the shRNAs significantly inhibited pSTAT3 to the same extent. This indicates that total knockdown (KD) of HIP1 is not necessary to completely shut-down the activity of pSTAT3. How does this translate to the biological functions of HIP1?
How come DMSO treatment blocks the phosphorylation of ERK1/2 in lane 2 of Fig 1(F)?
Figure S1F: pSTAT3 western blot: the authors should indicate which band they considered positive for p-STAT3; if it's the lower band why was there no activity in lane 4?
Fig 2A and 2B should be repeated in HIP1 knockout cells.
What is the endogenous level of HIP1 and GDF15 in prostate cancer cell lines vs. normal prostate epithelial cells? Why was HIP1 overexpressed in LNCaP cells? Was the level of HIP1 expression low in LNCaP and PNT1A, when compared in a panel of prostate cancer cell lines? Did the authors observe any differential expression of HIP1 and GDF15 in hormone sensitive vs. hormone resistant prostate cancer cells?
GDF15 is a very ambiguous biomarker of cancer as its levels are even higher in the case of mental disorders including psychosis (for reference https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554200/ ). And from this study, it is not even clear that the GDF15 upregulation is just one of the several outcomes of the activation of this signaling axis or if it is the only consequence of this signaling axis to promote the growth of cancer cells by increasing paracrine signaling. An experiment in GDF15 knockout cells/mice can document the role of this axis in a more precise manner.
It has been shown that wt p53 significantly reduces STAT3 tyrosine phosphorylation and inhibits STAT3 DNA binding activity in prostate cancer cell lines that express both constitutively active STAT3 and mutant p53 protein. The authors have claimed that the increase in STAT3 phosphorylation is due to HIP1 expression. All three of the cell lines evaluated in this paper have different p53 status and show differences in expression of activated STAT3. Is the expression of HIP1 independent of the status of p53?
Figure 3: Does STAT3 silencing (siRNA/stattic) downregulate HIP1, and does this decrease STAT3 activation over time? Also, does STAT3 silencing or treatment with WP1066 inhibit HIP1-induced tumor growth in vivo?
The role of GDF15 in prostate cancer is likely stage specific. It may promote early stages of tumorigenesis, but suppress the progression of advanced prostate cancers. The authors claim that HIP1 overexpression is mediated by stat3 activation, which leads to increased secretion of GDF15. Does expression of HIP1 correlate with the expression of GDF15 and does this also associate with stage-specific progression of prostate cancer?
How was cellular transformation studied and confirmed? Did HIP1 cause transformation of normal prostate cells?
Fig 1B: HIP1 western blot is not clear, please quantify 1C, 1D, 1E.
Most of the studies are done only in one cell line which is not adequate.
What is the clinical relevance of this study? The authors should study clinical samples along with multiple cell lines.
Several of the Western blot figures need better quality blots; Figs 1E (FGFR), S2C (all).
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###Reviewer #2:
The paper describes a novel signaling pathway which links HIP1 and STAT3. HIP1 is an oncolgene which should be targeted in prostate cancer. In previous studies the role of HIP1 in prostate cancer was established. The paper is well-written and the experiments needed to make appropriate conclusions are performed. The paper is also important because of identification of the role of GDF15 in prostate cancer. In my opinion, the paper may benefit from clarification whether HIP1 treatment leads to up-regulation of cytokines such as interleukin-6. This is possible because the effect of HIP1 could also be indirect, i.e. mediated by interleukin-6. No other major revisions are suggested. In general, the paper is an important contribution to understanding of signaling pathways of STAT3 in prostate cancer.
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###Reviewer #1:
In this manuscript by Rao et al, the authors use an immortalized prostate cancer epithelial cell line, PNT1A, to identify the effects of HIP1 overexpression. The authors show in a series of well-controlled experiments the positive relationship between HIP1, phosphorylation of STAT3, and expression of FGFR4. Phenotypically, this relationship is also associated with pro-tumorigenic events such as in vitro migration and invasion, and development of tumor xenografts. Finally, the authors demonstrate that HIP1 results in increased expression of the GDF15 cytokine to exert its effects on tumor cells in a paracrine fashion.
There are no major concerns with this manuscript.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
In this manuscript by Rao et al, the authors use an immortalized prostate cancer epithelial cell line, PNT1A, to identify the effects of HIP1 overexpression. The authors define a positive relationship between HIP1, phosphorylation of STAT3, and expression and activation of the FGF2 receptor, FGFR4. Phenotypically, this relationship is also associated with pro-tumorigenic events such as in vitro migration and invasion, and growth of tumor xenografts. Finally, the authors make the case that HIP1 results in …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
In this manuscript by Rao et al, the authors use an immortalized prostate cancer epithelial cell line, PNT1A, to identify the effects of HIP1 overexpression. The authors define a positive relationship between HIP1, phosphorylation of STAT3, and expression and activation of the FGF2 receptor, FGFR4. Phenotypically, this relationship is also associated with pro-tumorigenic events such as in vitro migration and invasion, and growth of tumor xenografts. Finally, the authors make the case that HIP1 results in increased expression of the GDF15 cytokine to exert its effects on tumor cells in a paracrine fashion.
In general, the paper is well-written, and the results clearly presented. The authors have previously extensively studied HIP1 in cancer, including prostate cancer (Rao et al., 2002). A role for HIP1 in STAT3 signaling has also been demonstrated (Hsu et al, 2015). Hence, the primary novelty and importance of the study is because of identification of role of GDF15 in prostate cancer, and delineation of a tumor-promoting, paracrine HIP1-STAT3-GDF15 signaling axis. While this was viewed as a strength of the study, there were significant weaknesses. Most prominent of the weaknesses was the fact that the bulk of the experiments were performed only in a single cell model, PNT1A, which reduces confidence that the results are generalizable, as opposed to reflecting idiosyncratic signaling response in this model. The consensus of the reviewers was that the key findings of the studies should be further validated in additional cell line models, and/or the relationships proposed should be validated in clinical specimens for prostate cancer. Ideally, both additional cell lines and clinical samples would be used, but at least one is essential to support conclusions. In addition to this important global critique, the reviewers made several specific criticisms of the experiments presented in the study, which should be addressed.
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