Opposite changes in the expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue: putative clathrin-mediated recycling of EGFR

doi:10.21203/rs.3.rs-1503162/v1

Download PDF

Research Article

Opposite changes in the expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue: putative clathrin-mediated recycling of EGFR

https://doi.org/10.21203/rs.3.rs-1503162/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 04 Mar, 2023

Read the published version in Histochemistry and Cell Biology →

You are reading this latest preprint version

Endocytosis, an important macromolecule uptake process in cells, is known to be dysregulated in cancer. Clathrin and caveolin-1 proteins play a major role in receptor-mediated endocytosis. We have used a quantitative, unbiased and semi-automated method to measure in situ protein expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue. There was a significant (p < 0.0001) increase in the expression of clathrin in prostate cancer samples (N = 29, n = 91) compared to normal tissue (N = 29, n = 67). Conversely, there was a significant (p < 0.0001) decrease in expression of caveolin-1 in prostate cancer tissue compared to normal prostate tissue. The opposite change in expression of the two proteins was highly correlated to increasing cancer aggressiveness. There was also a concurrent increase in the expression of epidermal growth factor receptor (EGFR), a key receptor in carcinogenesis, with clathrin in prostate cancer tissue, indicating recycling of EGFR through clathrin-mediated endocytosis (CME).

These results indicate that in prostate cancer, caveolin-1-mediated endocytosis (CavME) may be acting as a brake and increase in CME may facilitate tumorigenicity and aggressiveness of prostate cancer through recycling of EGFR. Changes in the expression of these proteins can also potentially be used as a biomarker for prostate cancer to aid in diagnosis and prognosis and clinical decision-making.

Prostate

Cancer

Clathrin

Caveolin

EGFR

Endocytosis

Receptor-mediated endocytosis (RME) allows internalization of specific macromolecules by a cell from the extracellular space by first binding to a specific cell receptor found in clathrin-coated pits of the cell membrane [reviewed in (Khan & Steeg, 2021)]. Clathrin, so named to indicate a lattice-like structure (Pearse, 1976) forms a triskelion shape made up of three heavy and three light chains with two isoforms identified for each chain (Brodsky, 1985; Kaksonen & Roux, 2018). The clathrin pits bud to form clathrin-coated vesicles with the bound receptor ligand complex and the process is termed clathrin-dependent or clathrin-mediated endocytosis (CME). CME has been known to play a key role in cancer and clathrin is overexpressed in lung, liver and ovarian cancer (Seimiya et al., 2008; Smith et al., 2013; Wei et al., 2019).

In addition to CME there are other, clathrin-independent endocytotic processes, such as caveolae-dependent endocytosis (CavME), which is driven by integral membrane proteins called caveolins (Rothberg et al., 1992). Caveolae are small (50-100nm) plasma membrane invaginations containing oligomeric integral membrane proteins called caveolins and peripheral membrane proteins collectively termed cavins (Hansen & Nichols, 2010; Yamada, 1955). Three isoforms of caveolins, ranging in molecular weight from 18-22kDa, encoded by CAV1, 2 and 3 genes, are known. In addition to their role in clathrin-independent endocytosis, caveolins are also involved in intracellular Ca²⁺ signaling and mechano-transduction (Del Pozo et al., 2021; Gilbert et al., 2016). Both CME and CavME have been shown to play an important role in carcinogenesis and cancer metastasis. It has been suggested that the deregulation of endocytosis is likely to be a hallmark in many cancers (Mellman & Yarden, 2013).

Prostate cancer is the second leading cancer in men, worldwide (Ferlay et al., 2021), it is curable when confined to the organ but is a major cause of mortality when metastasized. It is known that dysregulation of receptor-mediated cell signaling pathways may play a role in both carcinogenesis and metastasis of prostate cancer (Baetke et al., 2012; Murillo-Garzon & Kypta, 2017). There is, however, patchy information regarding the expression of clathrin or caveolin-1 in human prostate tissue. There are some studies of caveolin expression in human prostate tissue showing association of caveolin-1 with cancer progression and recurrence (Karam et al., 2007; Satoh et al., 2003; Yang et al., 2005; Yang et al., 1998). There is also some evidence from investigations of mechanisms in cell lines. For example, Prescott and Tindall (1998) observed androgen-induced clathrin mRNA and protein overexpression in LnCaP prostate cancer cell line (Prescott & Tindall, 1998). It is thought that at a cellular level, clathrin also promotes prostate cancer cell proliferation and migration by binding to cadherin-11 and prostate-specific membrane antigen (Goodman et al., 2007; Satcher et al., 2015).

Extracellular ligand and membrane bound receptor-dependent cell signaling pathways such as EGFR, Wnt, Notch, Hedgehog, and NF-κB are critical during the process of carcinogenesis and metastasis (Nwabo Kamdje et al., 2017; Yang et al., 2020). Endocytosis is a major regulator for receptor availability for these signaling pathways. Receptor availability on the cell membrane can enhance or reduce the activity of key proliferative pathways [1, 13]. If RME instead of degradation, recycles the receptor this may result in increased cell proliferation or carcinogenesis. There is evidence that EGFR is subject to such a regulation via CME (Chi et al., 2011). However, little information regarding the role of endocytosis-mediated receptor regulation for carcinogenic pathways such as EGFR exist, particularly in human tissue and in prostate cancer.

Furthermore, CME and CavME, like many other cellular processes, function in parallel and it is therefore important to investigate the relationship between the expression of clathrin and caveolin-1, simultaneously, in human prostate cancer tissue. In order to interrogate the molecular basis of CME and CavME in prostate cancer, we have used a multi-label immunohistochemical technique, combined with unbiased quantitative analysis (Arthurs et al., 2017; Symes et al., 2013), to simultaneously measure the expression of clathrin and caveolin-1, in normal and paired cancerous prostate tissue with different Gleason grades on tissue arrays (TAs). We tested the hypothesis that RME is increased in prostate cancer and is correlated to cancer progression. We also hypothesized that there is increased recycling of EGFR in prostate cancer which may be correlated to CME. Our results show a concurrent and opposite change in the expression of these clathrin and caveolin-1 proteins in prostate cancer and as the cancer progresses towards aggressive disease. We further show that there is no change in the interaction between caveolin-1 and EFGR but there is a significant increase in EGFR colocalization with clathrin in prostate cancer tissue compared to normal. These results provide novel information regarding the expression and putative concurrent function of CME and CavME in prostate cancer development and progression. An important consequence increased CME may be increased carcinogenesis via receptor recycling for key signaling pathways such as EGFR.

Ethical approval and prostate tissue samples

Ethical approval (REC Ref 13/ES/0092) was granted by the joint research office University College London and University College Hospital through East of Scotland Research Ethics Service. Archival prostate tissue samples used primarily for pathological analysis, from Portuguese Oncology Institute of Porto (Porto, Portugal), were used to make tissue arrays utilized in this study. Samples were collected from patients with a median age of 64 (interquartile range of 61,68). Most recent PSA levels (ng/mL) at prostatectomy were also recorded as 9.30 (with an interquartile range 5.61,11.80). A total of 29 individual prostate tissue samples classified by an expert uropathologist as cancerous and further sub-classified for Gleason scores were used; 29 cancer adjacent, non-cancerous, tissue samples were also extracted. The experiment was conducted as a double blind study with neither the experimenter (who performed antibody staining) nor the data analysts being aware of identity of the condition of the tissue samples or that of the antibody used. The data was only decoded after the anonymized, quantitative analysis was completed (Symes et al., 2013).

TA construction

TA were constructed in our laboratory using a manual tissue arrayer (MTA1, Beechers Instruments, Sun Prairie, WI, USA) as described in detail elsewhere (Nariculam et al., 2009; Symes et al., 2013). Individual tissue cores (1mm in diameter), were extracted from the marked regions of prostate cancer and cancer adjacent (normal) prostate tissue blocks and moved to a recipient block using a Beechers MTA1 manual tissue arrayer (Beechers Instruments, Sun Prairie, WI, USA). Five recipient blocks were populated with samples (five to six tissue samples) from both prostate cancer (N=29, n=91) cancer adjacent region (N=29; n=67) of the whole tissue blocks (N= the number of patients, n= number of cores). The following distribution of Gleason grades were used: 3+3 (n=15); 3+4/4+3 (n=11); 4+4 (n=43); and 4+5/5+4 (n=22).

TA blocks were sectioned at 6-8μm thickness on glass slides using a Thermo Scientific^TM HM355S automatic microtome. Sections on slides were stained with Hematoxylin and Eosin (H&E) and imaged at 40x magnification using NanoZoomer Digital Pathology Scanner 2.0 RS (Hamamatsu, Hertfordshire, UK) and re-examined by the pathologist.

Immunohistochemistry

Multiplex staining of TAs, using anti-clathrin (Abcam, Cat. No. ab21679), anti-caveolin-1 (Abcam, Cat. No. ab2910) and EGFR (Leica, NCL-EGFR) antibodies on the same TA section, was conducted using Bond Max automated staining system (Leica, UK) (Arthurs et al., 2020; Symes et al., 2013). Each antibody was optimized for pH and concentration dependence, antigen retrieval and temperature parameters; nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Antibodies were used at a dilution of 1:2000, 1:4000 and 1:500 for anti-clathrin, anti-caveolin-1 and EGFR, on TAs and labelled with Opal-650 (red), Opal-520 (green) and Opal-570 (yellow), respectively.

Imaging of immunofluorescently labelled TAs

Tissue cores from multi-labelled immunofluorescence TAs were imaged using AxioScan Z1 scanner (Zeiss, Cambridge, UK) at 20x magnification for protein expression analysis, as described previously (Arya et al., 2015). The excitation/emission setting of different channels were 353/465nm (DAPI); 493/517nm (caveolin-1); 653/668nm (clathrin) and 550/570nm (EGFR). Fluorescent signals were optimized to avoid saturation and for comparative analysis. The power settings and integration times of Hamamatsu ORCA Flash4 camera (Hamamatsu Photonics, Japan) on AxioScan were kept constant for all tissue cores imaged. For standardization, all imaging was conducted on the same day with all other image settings kept the same. Images of individual tissue cores (scenes in AxioScan Z1) were extracted from multiple 5712x5712 pixel, 300dpi, 24-bit single color images and stored as tiff files.

For the measurement of colocalization coefficients (Global Intersection Coefficient, GIC) between different fluorescent labels, high magnification confocal imaging on a Leica SP8 microscope (Leica, UK) with a 63x 1.4-NA oil objective and 6x optical zoom at a 1024x1024 pixel format, was also conducted on sub-sets of different prostate tissue samples. The Z-stack scan was performed at 0.17μm (at 600Hz), yielding approximately 26-30 Z-sections for each tissue core.

Quantitation of clathrin and caveolin-1 signal in prostate tissue

The expression levels for clathrin and caveolin-1 were quantified using an unbiased, quantitative analysis developed in our laboratory (Arya et al., 2015; Symes et al., 2013) using an adapted ImageJ plugin (Rasband, 2011). Briefly, the images were separated into red (Opal 650, clathrin), green (Opal 520, caveolin-1) and yellow (Opal-570, EGFR) channels and were exported from ZEN 3.1 (blue edition) software, opened in ImageJ software and were converted to 8-bit greyscale; a threshold and segmentation routine was then applied. Lower and upper threshold parameters were set to include the median to maximum grey values of a range of samples across the sample cohort. The grey values were then used to perform semi-automated analysis of the whole tissue array. A similar approach was implemented to quantify the amount of tissue in each core and this value was divided by the intensity to obtain a standardized signal for each label in prostate tissue samples.

Statistical analysis was performed using MedCalc software (version 19.7.2, www.medcalc.org) using Mann-Whitney U test and boxplots were made using both GraphPad Prism 9 software (GraphPad Software, CA, USA) and Origin pro software (OriginLab, MA, USA).

Deconvolution and colocalization analysis using high magnification confocal images

158 individual tissue cores (67 normal, 91 malignant cores including 15 Gleason score 3+3, 11 Gleason score 3+4, 43 Gleason score 4+4 and 22 Gleason score 4+5 with at least 4 ROIs for each core) were identified for areas of cancer visually on the serial H&E section of the tissue array. These areas were approximated on the fluorescently labelled tissue array and tissue cores were imaged using a Leica SP8 confocal system as described above.

The resulting image files were deconvolved using Huygens Professional software (version 20.04, Netherlands). Deconvolved images were saved as Huygens specific (HDF5) files for each channel (excitation/emission (nm) DAPI 405 / 429, Opal-520 = 488/517, Opal-570 = 561 / 595 and Opal-650 = 635 / 668). The HDF5 files were imported into Huygens co-localization module and were background subtracted using ‘Gaussian minimum’ method; up to 4-16 regions of interest/image were analyzed to calculate the global intersection coefficient for co-localization (Arthurs et al., 2020). Colocalization analysis was conducted using Huygens professional software to calculate GICs.

Histopathological analysis

An expert uropathologist diagnosed and marked the tissue sections (Supplementary Fig. 1) from which normal (cancer adjacent, non-cancerous) and cancerous prostate tissue were extracted for making tissue arrays. Subsequent to tissue array construction, the cores were verified and validated for diagnosis, including grading in each core, using H&E sections (Supplementary Fig. 2).

Comparison of expression of clathrin and caveolin-1 in normal (cancer adjacent, non-cancerous) and cancer prostate tissue

Tissue arrays were probed, simultaneously, with anti-clathrin and anti-caveolin-1 antibodies; both these proteins were found to be expressed in prostate tissue (Fig. 1). There was a visible increase in the expression of clathrin in cancerous prostate compared to normal. Representative tissue cores for normal and cancer prostate tissue with clathrin (red) and caveolin-1 (green) show an increase in the expression of the former and corresponding decrease of expression of the latter in cancer tissue (Fig. 1). We used an ImageJ based image quantitation method, developed in our laboratory (Arya et al., 2015), to quantify the expression signal for clathrin and caveolin-1. What was seen by eye (Fig. 1) was confirmed upon quantitation (Fig. 2a, b and c).

When a normal vs. cancer comparison was made for the expression of two proteins, there was a significant (p<0.0001, Mann-Whitney) 2.5 fold increase in the expression of clathrin in prostate cancer tissue compared to corresponding controls (Fig. 2a); also, there was a concurrent and significant, ~2.2 fold (p<0.0001) decrease in the expression of caveolin-1 in prostate cancer tissue compared to normal tissue (Fig. 2b). The interquartile range of the expression of clathrin and caveolin-1 is shown in Fig. 2c. These results show, for the first time, that as there is an increase of clathrin protein expression in prostate cancer tissue, there is a concurrent, significant and opposite decrease in the expression of caveolin-1.

Clathrin and caveolin-1 expression in low and high grade prostate cancer

Prostate tissue used in the analysis above (Supplementary Fig. 1 and Supplementary Fig. 2, Fig. 1 and 2) contained cancer samples of different Gleason grades (see also Table S1) that characterize the diagnosis of prostate cancer. Fig. 3 shows a selection of prostate tissue used in this study probed with anti-clathrin (red) and anti-caveolin-1 (green) antibodies. There is a gradual increase in the expression of clathrin from normal, low grade (3+3 and 3+4/4+3) to high grade (4+4 and 4+5/5+4) prostate cancer. Although clathrin appears to be expressed mostly in the epithelium, caveolin-1 can be seen both in the stroma and in the acinar epithelium. Images including the ones shown here were used to quantify the expression of clathrin and caveolin-1 in different Gleason grade samples (Fig. 4).

Clathrin was significantly overexpressed in different Gleason grades of prostate cancer except 3+4/4+3 (Fig. 4a). For caveolin-1, although there was no significant difference between normal and Gleason 3+3 group (p>0.05), there was a significant decrease in caveolin-1 expression with increasing Gleason score: Gleason 3+4/4+3 (p<0.002); Gleason 4+4 (p<0.0001) and Gleason 4+5/5+4 (p<0.0001) (Fig. 4b).

Segregating samples according to different Gleason scores and comparing these against normal tissue shows that (i) clathrin expression shows a decrease in score 3+4 and 4+4 but the expression reverts back up to the levels of 3+3 Gleason grade in high Gleason grade cancer (Fig. 4a) and (ii) there is a gradual and persistent decrease in the expression of caveolin-1 with increasing Gleason score (Fig. 4b). This indicates that prostate cancer progression from low Gleason grade may require increase in clathrin and by extension CME which may again be necessary for pre-metastatic cancer. Noting that there is a general decrease in caveolin-1 expression compared to normal prostate tissue (Fig. 2b), these results suggest that caveolin-1 plays a key role in early carcinogenesis process, perhaps as a suppressor of tumorigenesis. These results show that in prostate cancer, the combination of a gradual decrease in caveolin-1 and increase in clathrin expression cancer may be critical for cancer progression.

Clathrin and caveolin-1 expression in cancers with different Gleason scores

In addition to the comparison between normal and cancer tissue, we also compared the expression of clathrin and caveolin-1 in cancers diagnosed with different Gleason scores (Fig. 4c and d). Clathrin expression appears to decrease initially but reverts to low grade cancer (3+3) as the cancer reaches aggressive phenotypes (4+5) (Fig. 4c). Conversely, caveolin-1 levels appear to decrease in low grade cancer (3+3) compared to normal (Fig. 4b) and from then on show a consistent and significant decline as the aggressiveness increases (Fig. 4d). These results further support the notion of counter balancing roles of CavME vs. CME in the development and progression of prostate cancer.

Colocalization analysis of clathrin and caveolin-1 in normal and cancerous prostate tissue

We have previously shown that in addition to protein expression, colocalization of different proteins may serve as an additional differential parameter and biomarker between non-cancerous and cancerous tissue samples (Arthurs et al., 2020). There are no reports regarding colocalization of clathrin and caveolin-1 in human prostate tissue or whether these are closely or distantly localized? To measure the colocalization of these two proteins, we imaged the tissue cores at a high magnification (x63) using confocal microscopy. Fig. 5 shows tissue cores at low magnification (20x, Fig. 5a and b) and areas of the same cores (see methods) imaged at 63x magnification (Fig. 5c and d). Deconvolution analysis using Huygens (SVI) deconvolution software was performed on high magnification, high resolution confocal images for the calculation of the GIC that estimates the proximity of pixels for two channels (Fig. 5e and f). We also used DAPI, to stain the nuclei and estimate the colocalization of clathrin and caveolin-1 with nuclei (Fig. 5g). There was no significant difference in the colocalization of clathrin and caveolin-1 in normal compared to cancer prostate tissue samples (Fig. 5g). Furthermore, there was no difference between the GIC for clathrin and caveolin-1 across tumors with different Gleason scores (Supplementary Fig. 3). However, our results show a significant (p=0.02) increase in the colocalization between clathrin and DAPI (a nuclear marker) in cancerous compared to normal tissue (Fig. 5e). The GIC for clathrin and DAPI increased in Gleason score 3+3 and 4+4 cancer tissue compared to normal (Supplementary Fig. 3). This indicates that clathrin may influence transcriptional processes during carcinogenesis. Conversely, decreased caveolin-1/DAPI GIC values was found in cancer compared to normal (p=0.03), indicating a loss of caveolin expression in the nucleus in cancer. This further suggests a putative tumor suppressor role of caveolin-1, however, it is only indicative and would require further investigations to show a role in transcription in prostate cancer for either clathrin or caveolin-1. GIC for various permutations of labels between normal and cancerous tissue with different Gleason scores are shown in Supplementary Fig. 3.

Clathrin, caveolin-1 and EGFR in prostate cancer

To test the hypothesis that an important mechanism in prostate carcinogenesis is the receptor recycling for key cell proliferative pathways such as EGFR (Sigismund et al., 2008), we conducted simultaneous, multi-labelled staining of clathrin, caveolin-1 and EGFR. Fig. 6 shows a normal and a cancer tissue core with combined clathrin/EGFR (Fig. 6a and b) and caveolin-1/EGFR channels (Fig. 6c and d). The expanded, high-magnification images from an area of the cores are shown in Fig. 6 e-h. Deconvolved images, subsequent to high-resolution confocal microscopy, were used to measure GIC in prostate normal and cancer tissue (Fig. 6i-l). Quantitation of GIC between normal and cancer in two protein pairs (clathrin/EGFR, caveolin-1/EGFR) is also shown (Fig. 6m); single-channel images are shown in Supplementary Fig. 4. These results indicate a significant increase of the colocalization between clathrin and EGFR in cancerous tissue compared to normal (p<0.0001) (Fig. 6m), indicating that clathrin may play an important role in facilitating EGFR recycle via CME process.

In this investigation we show for the first time, an opposite expression of the proteins involved in two key endocytotic pathways, namely, CME and CavME. We demonstrate that there is an increase in the expression of clathrin with increasing Gleason score in prostate cancer and a concurrent decrease in caveolin-1 expression. The expression of clathrin is almost exclusively found in epithelial cells in both normal and cancer samples whereas caveolin-1, in addition to being expressed largely in stromal tissue, also shows expression in epithelial cells (Fig. 1, 3, 5 and 6) in normal tissue. The overall increase in the expression of clathrin in prostate cancer (Fig. 2) could be explained by the increase in the number of epithelial acini as the cancer progresses. A simple correlative conclusion of these results appears to be that caveolin-1 expression and by extension CavME may act as a brake on the tumorigenesis; decrease in caveolin-1 expression may accelerate tumorigenesis which is further enhanced by over-expression of clathrin with its known function in receptor recycling for key carcinogenic pathways (Imbastari et al., 2021).

Indeed, an interesting aspect of CME and clathrin independent endocytosis is the targeted internalization of key receptor signaling involved in cell proliferation (Brunt & Scholpp, 2018; Perez Verdaguer et al., 2021; Wee & Wang, 2017). In the context of receptor-mediated cell signaling, endocytosis may (i) attenuate an extracellular signal (ii) or facilitate signal reactivation via receptor recycling back to the cell surface. We discuss some of the ramifications of our results in prostate cancer and provide a framework for the putative mechanisms that may also be relevant to other cancers.

CME activates proliferative signaling pathways

Cancer metastasis is a complex, incremental process, partly a result of interaction between tumor cells and their microenvironment. Endocytosis is a major regulator for receptor availability for these signaling pathways (Khan & Steeg, 2021; Mellman & Yarden, 2013). As a result, there is emerging interest in endocytosis as a key process associated with carcinogenesis, reduced tumor suppression and metastasis in different cancer types (Mellman & Yarden, 2013). We discuss these notions within the framework of two key carcinogenic EGFR signaling. Various elements of the Wnt signaling are known to be mutated in epithelial cancers, including those of the prostate. Furthermore, EGFR signaling has been subject of many investigations with regards to the role of RMEs (Khan & Steeg, 2021).

Proliferative signaling processes, such as the EGFR pathway, also require endocytosis for signal activation that result in increased transcriptional activity of proto-oncogenes. It has been suggested that nearly 20% of all prostate cancers may have activated EGFR signaling (Hashmi et al., 2019). An interesting mechanism of CME, proposed by Sigismund and colleagues (Sigismund et al., 2008), is that EGFR is internalized by CME but rather than being degraded, these receptors are recycled to the cell membrane. Our results suggest that in prostate cancer, increase in clathrin expression is associated with an increased colocalization with EGFR (Fig. 6), indicating EGFR recycling may allow cancer to progress through a constitutive proliferative signaling mechanism.

Other signaling pathways such as the Wnt signaling network also play an important role in carcinogenesis and cancer metastasis (Murillo-Garzon & Kypta, 2017). Wnt proteins are upregulated in prostate and other cancers (Wang et al., 2010; Yang et al., 2006) and play a key role in cell proliferation. Wnt ligands (e.g. Wnt3A, 4, 5A, 7, 9B) act as paracrine signals, produced from source cells and act on their target cells (Brunt & Scholpp, 2018). It is known that Wnt signaling is under the regulation of CME (Blitzer & Nusse, 2006). CME of Wnt/Fzd receptors is thought to enhance Wnt/β-catenin signal (Brunt & Scholpp, 2018), although how endocytosis regulates or regulated by Wnt signaling is not clear. However, the mechanism of CME regulation of Wnt signaling is different from that proposed for other signaling mechanisms such as EGFR – unlike the receptor recycling for EGFR activation by CME (see below), endocytosis of Wnt ligands occurs in the source cells (Brunt & Scholpp, 2018; Sigismund et al., 2018). A further interesting aspect of endocytosis control of Wnt signaling is the suggestion that Wnt3A-LRP6 receptor complex internalization by CavME. Caveolin-1 is also reduced in other cancers such as those of the breast, liver and ovary [reviewed in (Goetz et al., 2008)]. Considering the role of CME and CavME in EGFR and Wnt signaling, our data (Fig. 2 and 3) yields an interesting hypothesis: the decline in the expression of caveolin-1, may be acting as a tumor suppressor whereas clathrin overexpression in progressive grades of prostate cancer may suggest a tumor promotion role through its regulation of Wnt signaling. A similar role could also be construed for Erb2 signaling (Hynes & MacDonald, 2009).

In summary, we present data that suggests a novel role of endocytotic pathways in human prostate cancer. Our results indicate that CME and CavME may act in concert in prostate carcinogenesis and progression. These results highlight the central role of endocytosis in prostate cancer and suggest the possibility of endocytosis pathways as a disease stratification biomarker and a target for therapy of prostate cancer.

Acknowledgements: We are grateful to Prostate Cancer Research Centre charity for partly supporting this project. We are also grateful to the King’s College Development Fund for financial support.

Clinical trial registration: not applicable

Conflict of interest disclosure: The authors have no relevant financial or non-financial interests to disclose.

Data availability: All data is available through supplementary material

Ethics approval statement: Ethical approval was granted by the joint research office University College London and University College Hospital through East of Scotland Research Ethics Service (REC Ref 13/ES/0092).

Funding: The research was partly funded by a grant award from Prostate Cancer Research Centre charity and King’s College Development Fund to AA.

Patient consent statement: Only archival, anonymized tissue samples were used.

Permission to reproduce material from other sources: not applicable

Author contributions statement

B.X., H.Z., M.M., T.R., C.T, and K.D. conducted experiments; B.X., H.Z., C.T., J.P., and A.A. analyzed data; R.H, collected and characterized the tissue samples; A.M., and H.P. provided clinical input; B.X., and A.A. wrote the manuscript; H.Z., R.H., M.M., T.R., C.T., K.D., J.P., A.M., and H.P. reviewed the manuscript; A.A. designed the study and supervised experimentation.

Arthurs, C., Murtaza, B. N., Thomson, C., Dickens, K., Henrique, R., Patel, H. R. H., Beltran, M., Millar, M., Thrasivoulou, C., & Ahmed, A. (2017). Expression of ribosomal proteins in normal and cancerous human prostate tissue. PLoS One, 12(10), e0186047. https://doi.org/10.1371/journal.pone.0186047
Arthurs, C., Suarez-Bonnet, A., Willis, C., Xie, B., Machulla, N., Mair, T. S., Cao, K., Millar, M., Thrasivoulou, C., Priestnall, S. L., & Ahmed, A. (2020). Equine penile squamous cell carcinoma: expression of biomarker proteins and EcPV2. Sci Rep, 10(1), 7863. https://doi.org/10.1038/s41598-020-64014-3
Arya, M., Thrasivoulou, C., Henrique, R., Millar, M., Hamblin, R., Davda, R., Aare, K., Masters, J. R., Thomson, C., Muneer, A., Patel, H. R., & Ahmed, A. (2015). Targets of Wnt/ss-catenin transcription in penile carcinoma. PLoS One, 10(4), e0124395. https://doi.org/10.1371/journal.pone.0124395
Baetke, S. C., Adriaens, M. E., Seigneuric, R., Evelo, C. T., & Eijssen, L. M. (2012). Molecular pathways involved in prostate carcinogenesis: insights from public microarray datasets. PLoS One, 7(11), e49831. https://doi.org/10.1371/journal.pone.0049831
Blitzer, J. T., & Nusse, R. (2006). A critical role for endocytosis in Wnt signaling. BMC Cell Biol, 7, 28. https://doi.org/10.1186/1471-2121-7-28
Brodsky, F. M. (1985). Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites. J Cell Biol, 101(6), 2047–2054. https://doi.org/10.1083/jcb.101.6.2047
Brunt, L., & Scholpp, S. (2018). The function of endocytosis in Wnt signaling. Cell Mol Life Sci, 75(5), 785–795. https://doi.org/10.1007/s00018-017-2654-2
Chi, S., Cao, H., Wang, Y., & McNiven, M. A. (2011). Recycling of the epidermal growth factor receptor is mediated by a novel form of the clathrin adaptor protein Eps15. J Biol Chem, 286(40), 35196–35208. https://doi.org/10.1074/jbc.M111.247577
Del Pozo, M. A., Lolo, F. N., & Echarri, A. (2021). Caveolae: Mechanosensing and mechanotransduction devices linking membrane trafficking to mechanoadaptation. Curr Opin Cell Biol, 68, 113–123. https://doi.org/10.1016/j.ceb.2020.10.008
Ferlay, J., Colombet, M., Soerjomataram, I., Parkin, D. M., Pineros, M., Znaor, A., & Bray, F. (2021). Cancer statistics for the year 2020: An overview. Int J Cancer. https://doi.org/10.1002/ijc.33588
Gilbert, G., Ducret, T., Savineau, J. P., Marthan, R., & Quignard, J. F. (2016). Caveolae are involved in mechanotransduction during pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol, 310(11), L1078-1087. https://doi.org/10.1152/ajplung.00198.2015
Goetz, J. G., Lajoie, P., Wiseman, S. M., & Nabi, I. R. (2008). Caveolin-1 in tumor progression: the good, the bad and the ugly. Cancer Metastasis Rev, 27(4), 715–735. https://doi.org/10.1007/s10555-008-9160-9
Goodman, O. B., Jr., Barwe, S. P., Ritter, B., McPherson, P. S., Vasko, A. J., Keen, J. H., Nanus, D. M., Bander, N. H., & Rajasekaran, A. K. (2007). Interaction of prostate specific membrane antigen with clathrin and the adaptor protein complex-2. Int J Oncol, 31(5), 1199–1203. https://www.ncbi.nlm.nih.gov/pubmed/17912448
Hansen, C. G., & Nichols, B. J. (2010). Exploring the caves: cavins, caveolins and caveolae. Trends Cell Biol, 20(4), 177–186. https://doi.org/10.1016/j.tcb.2010.01.005
Hashmi, S. K., Irfan, M., Asif, H., Nisar, L., Naeem, M., Khan, E. Y., Baloch, S., & Faridi, N. (2019). Prognostic utility of epidermal growth factor receptor (EGFR) expression in prostatic acinar adenocarcinoma. Applied Cancer Research, 39(1), 1–8. https://doi.org/10.1186/s41241-018-0069-5
Hynes, N. E., & MacDonald, G. (2009). ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol, 21(2), 177–184. https://doi.org/10.1016/j.ceb.2008.12.010
Imbastari, F., Dahlmann, M., Sporbert, A., Mattioli, C. C., Mari, T., Scholz, F., Timm, L., Twamley, S., Migotti, R., Walther, W., Dittmar, G., Rehm, A., & Stein, U. (2021). MACC1 regulates clathrin-mediated endocytosis and receptor recycling of transferrin receptor and EGFR in colorectal cancer. Cell Mol Life Sci, 78(7), 3525–3542. https://doi.org/10.1007/s00018-020-03734-1
Kaksonen, M., & Roux, A. (2018). Mechanisms of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol, 19(5), 313–326. https://doi.org/10.1038/nrm.2017.132
Karam, J. A., Lotan, Y., Roehrborn, C. G., Ashfaq, R., Karakiewicz, P. I., & Shariat, S. F. (2007). Caveolin-1 overexpression is associated with aggressive prostate cancer recurrence. Prostate, 67(6), 614–622. https://doi.org/10.1002/pros.20557
Khan, I., & Steeg, P. S. (2021). Endocytosis: a pivotal pathway for regulating metastasis. Br J Cancer, 124(1), 66–75. https://doi.org/10.1038/s41416-020-01179-8
Mellman, I., & Yarden, Y. (2013). Endocytosis and cancer. Cold Spring Harb Perspect Biol, 5(12), a016949. https://doi.org/10.1101/cshperspect.a016949
Murillo-Garzon, V., & Kypta, R. (2017). WNT signalling in prostate cancer. Nat Rev Urol, 14(11), 683–696. https://doi.org/10.1038/nrurol.2017.144
Nariculam, J., Freeman, A., Bott, S., Munson, P., Cable, N., Brookman-Amissah, N., Williamson, M., Kirby, R. S., Masters, J., & Feneley, M. (2009). Utility of tissue microarrays for profiling prognostic biomarkers in clinically localized prostate cancer: the expression of BCL-2, E-cadherin, Ki-67 and p53 as predictors of biochemical failure after radical prostatectomy with nested control for clinical and pathological risk factors. Asian J Androl, 11(1), 109–118. https://doi.org/10.1038/aja.2008.22
Nwabo Kamdje, A. H., Takam Kamga, P., Tagne Simo, R., Vecchio, L., Seke Etet, P. F., Muller, J. M., Bassi, G., Lukong, E., Kumar Goel, R., Mbo Amvene, J., & Krampera, M. (2017). Developmental pathways associated with cancer metastasis: Notch, Wnt, and Hedgehog. Cancer Biol Med, 14(2), 109–120. https://doi.org/10.20892/j.issn.2095-3941.2016.0032
Pearse, B. M. (1976). Clathrin: a unique protein associated with intracellular transfer of membrane by coated vesicles. Proc Natl Acad Sci U S A, 73(4), 1255–1259. https://doi.org/10.1073/pnas.73.4.1255
Perez Verdaguer, M., Zhang, T., Paulo, J. A., Gygi, S., Watkins, S. C., Sakurai, H., & Sorkin, A. (2021). Mechanism of p38 MAPK-induced EGFR endocytosis and its crosstalk with ligand-induced pathways. J Cell Biol, 220(7). https://doi.org/10.1083/jcb.202102005
Prescott, J. L., & Tindall, D. J. (1998). Clathrin gene expression is androgen regulated in the prostate. Endocrinology, 139(4), 2111–2119. https://doi.org/10.1210/endo.139.4.5926
Rasband, W. S. (2011). Imagej, US National Institutes of Health. Bethesda, Maryland, USA. http://imagej.nih.gov/ij/
Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R., & Anderson, R. G. (1992). Caveolin, a protein component of caveolae membrane coats. Cell, 68(4), 673–682. https://doi.org/10.1016/0092-8674(92)90143-z
Satcher, R. L., Pan, T., Bilen, M. A., Li, X., Lee, Y. C., Ortiz, A., Kowalczyk, A. P., Yu-Lee, L. Y., & Lin, S. H. (2015). Cadherin-11 endocytosis through binding to clathrin promotes cadherin-11-mediated migration in prostate cancer cells. J Cell Sci, 128(24), 4629–4641. https://doi.org/10.1242/jcs.176081
Satoh, T., Yang, G., Egawa, S., Addai, J., Frolov, A., Kuwao, S., Timme, T. L., Baba, S., & Thompson, T. C. (2003). Caveolin-1 expression is a predictor of recurrence-free survival in pT2N0 prostate carcinoma diagnosed in Japanese patients. Cancer, 97(5), 1225–1233. https://doi.org/10.1002/cncr.11198
Seimiya, M., Tomonaga, T., Matsushita, K., Sunaga, M., Oh-Ishi, M., Kodera, Y., Maeda, T., Takano, S., Togawa, A., Yoshitomi, H., Otsuka, M., Yamamoto, M., Nakano, M., Miyazaki, M., & Nomura, F. (2008). Identification of novel immunohistochemical tumor markers for primary hepatocellular carcinoma; clathrin heavy chain and formiminotransferase cyclodeaminase. Hepatology, 48(2), 519–530. https://doi.org/10.1002/hep.22364
Sigismund, S., Argenzio, E., Tosoni, D., Cavallaro, E., Polo, S., & Di Fiore, P. P. (2008). Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev Cell, 15(2), 209–219. https://doi.org/10.1016/j.devcel.2008.06.012
Sigismund, S., Avanzato, D., & Lanzetti, L. (2018). Emerging functions of the EGFR in cancer. Mol Oncol, 12(1), 3–20. https://doi.org/10.1002/1878-0261.12155
Smith, C. M., Haucke, V., McCluskey, A., Robinson, P. J., & Chircop, M. (2013). Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells. Mol Cancer, 12, 4. https://doi.org/10.1186/1476-4598-12-4
Symes, A. J., Eilertsen, M., Millar, M., Nariculam, J., Freeman, A., Notara, M., Feneley, M. R., Patel, H. R., Masters, J. R., & Ahmed, A. (2013). Quantitative analysis of BTF3, HINT1, NDRG1 and ODC1 protein over-expression in human prostate cancer tissue. PLoS One, 8(12), e84295. https://doi.org/10.1371/journal.pone.0084295
Wang, Q., Symes, A. J., Kane, C. A., Freeman, A., Nariculam, J., Munson, P., Thrasivoulou, C., Masters, J. R., & Ahmed, A. (2010). A novel role for Wnt/Ca2 + signaling in actin cytoskeleton remodeling and cell motility in prostate cancer. PLoS One, 5(5), e10456. https://doi.org/10.1371/journal.pone.0010456
Wee, P., & Wang, Z. (2017). Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways. Cancers (Basel), 9(5). https://doi.org/10.3390/cancers9050052
Wei, X., Wei, R., Jiang, G., Jia, Y., Lou, H., Yang, Z., Luo, D., Huang, Q., Xu, S., Yang, X., Zhou, Y., Li, X., Ji, T., Hu, J., Xi, L., Ma, D., Ye, F., & Gao, Q. (2019). Mechanical cues modulate cellular uptake of nanoparticles in cancer via clathrin-mediated and caveolae-mediated endocytosis pathways. Nanomedicine (Lond), 14(5), 613–626. https://doi.org/10.2217/nnm-2018-0334
Yamada, E. (1955). The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol, 1(5), 445–458. https://doi.org/10.1083/jcb.1.5.445
Yang, G., Timme, T. L., Frolov, A., Wheeler, T. M., & Thompson, T. C. (2005). Combined c-Myc and caveolin-1 expression in human prostate carcinoma predicts prostate carcinoma progression. Cancer, 103(6), 1186–1194. https://doi.org/10.1002/cncr.20905
Yang, G., Truong, L. D., Timme, T. L., Ren, C., Wheeler, T. M., Park, S. H., Nasu, Y., Bangma, C. H., Kattan, M. W., Scardino, P. T., & Thompson, T. C. (1998). Elevated expression of caveolin is associated with prostate and breast cancer. Clin Cancer Res, 4(8), 1873–1880. https://www.ncbi.nlm.nih.gov/pubmed/9717814
Yang, L., Shi, P., Zhao, G., Xu, J., Peng, W., Zhang, J., Zhang, G., Wang, X., Dong, Z., Chen, F., & Cui, H. (2020). Targeting cancer stem cell pathways for cancer therapy. Signal Transduct Target Ther, 5(1), 8. https://doi.org/10.1038/s41392-020-0110-5
Yang, X., Chen, M. W., Terry, S., Vacherot, F., Bemis, D. L., Capodice, J., Kitajewski, J., de la Taille, A., Benson, M. C., Guo, Y., & Buttyan, R. (2006). Complex regulation of human androgen receptor expression by Wnt signaling in prostate cancer cells. Oncogene, 25(24), 3436–3444. https://doi.org/10.1038/sj.onc.1209366

No competing interests reported.

Download PDF

Journal Publication

published 04 Mar, 2023

Read the published version in Histochemistry and Cell Biology →

Editorial decision: Major revision
05 May, 2022
Reviews received at journal
20 Apr, 2022
Reviewers agreed at journal
08 Apr, 2022
Reviewers invited by journal
31 Mar, 2022
Editor assigned by journal
30 Mar, 2022
Submission checks completed at journal
30 Mar, 2022
First submitted to journal
29 Mar, 2022

You are reading this latest preprint version

Opposite changes in the expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue: putative clathrin-mediated recycling of EGFR

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Ethical approval and prostate tissue samples

TA construction

Immunohistochemistry

Imaging of immunofluorescently labelled TAs

Quantitation of clathrin and caveolin-1 signal in prostate tissue

Deconvolution and colocalization analysis using high magnification confocal images

Results

Histopathological analysis

Comparison of expression of clathrin and caveolin-1 in normal (cancer adjacent, non-cancerous) and cancer prostate tissue

Clathrin and caveolin-1 expression in low and high grade prostate cancer

Clathrin and caveolin-1 expression in cancers with different Gleason scores

Colocalization analysis of clathrin and caveolin-1 in normal and cancerous prostate tissue

Clathrin, caveolin-1 and EGFR in prostate cancer

Discussion

CME activates proliferative signaling pathways

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1