{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/372337"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/372337","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Investigating the role of the UDP-galactose transporter SLC35A2 in the regulation of HIF signalling","abstract":"Background: <br>The hypoxia inducible factor (HIF) family of dimeric transcription factors play a vital role in the cellular response to low oxygen (hypoxia). HIF activation leads to the transcriptional upregulation of a range of genes that are involved in diverse physiological and pathophysiological processes. In malignant tumours increased HIF-α expression, and HIF activation are commonly observed, and are associated with poorer prognosis and disease progression. HIF activity is controlled by a number of different mechanisms, and the identification of molecular regulators of HIF is of intense interest. The Golgi UDP-galactose transporter SLC35A2 was identified by the Ashcroft group as a novel regulator of HIF-α, and CHO cells that had lost SLC35A2 exhibited elevated HIF-α protein in normoxia. It is known that the absence of SLC35A2 in the Golgi leads to wide-ranging glycosylation defects. The underlying mechanisms linking SLC35A2 to HIF-α protein regulation were not known. This thesis aims to further characterise the role effect of SLC35A2 loss on HIF signalling and tumour cell behaviour, in CHO cells and a human cell system, specifically by exploring the hypotheses that these cells may harbour an autophagy defect, or differences in overall UDP-sugar content, in particular O-GlcNAc. Methods and Results: I used parental and Slc35a2 mutant CHO cells, SLC35A2 knockout (KO) and wild type (WT) suspension HeLa (sHeLa) cells, and a panel of cancer cells lines, including 786O, RCC4 renal carcinoma cells which exhibit constitutive HIF activation due to loss of VHL function. Confirming previous work from the Ashcroft group, I showed that Slc35a2 mutant (M6.19) CHO cells exhibit elevated normoxic HIF-1α protein levels and evidence of a glycosylation defect. Expanding on these findings, I found that SLC35A2 loss led to elevated normoxic HIF-α (HIF-1α and HIF-2α) protein and *HIFA* mRNΑ, and elevated expression of HIF-α target genes (*GLUT1*, *VEGF*). Using global RNAseq analysis of SLC35A2 KO and WT sHeLa cells, I found that SLC35A2 loss was associated with highly upregulated expression of a range of genes. Further exploration of the observed glycosylation defect in Slc35a2 mutant CHO cells, showed that SLC35A2 KO sHeLa cells also exhibited altered mobility of GLUT1 protein, which I found was consistent with treatment of WT sHeLa cells with tunicamycin, a protein *N*-glycosylation inhibitor. UDP-sugar analysis by HPLC of Slc35a2 mutant CHO and SLC35A2 KO sHeLa cells indicated increased levels of UDP-GlcNAc compared to their WT counterparts. Furthermore, I found similar patterns of increased UDP-GlcNAc levels in SLC35A2 KO compared to WT sHeLa cells in normoxia and hypoxia, while stable reconstitution of Slc35a2 in Slc35a2 mutant (M6.19) CHO cells reduced levels of UDP-GlcNAc and rescued CMP-sialic acid levels. Consistent with my findings and the involvement of UDP-GlcNAc in glutamine metabolism, I found that Slc35a2 mutant (M6.19) CHO cells were significantly more sensitive to glutamine withdrawal compared to parental (C4.5) CHO cells. Slc35a2 mutant (M6.19) and SLC35A2 KO sHeLa cells exhibited a glycosylation defect of LAMP2A, a protein involved in HIF-1α lysosomal degradation and critical for chaperone mediated autophagy. SLC35A2 mutant (M6.19) CHO cells were significantly more sensitive to the inhibition of proliferation by the inhibitors of autophagy bafilomycin and 3-methyladenine, and showed elevated levels of the autophagy marker LC3B. Finally, from analyses of the TCGA-KIRC database I found *SCL35A2* expression was higher in patients with non-mutant VHL versus mutant VHL in renal cancers. In support of these findings, I showed that basal SLC35A2 protein and mRNA levels were higher in patient-derived 786O renal carcinoma cells reconstituted with wild type VHL (786O-VHL) compared to matched 786-O empty vector control (786O-EV) cells. I also found that high *SLC35A2* expression is associated with poor prognosis in patients with *VHL* non-mutant ccRCC. Conclusion: Taken together, my thesis identifies SLC35A2 as a regulator of HIF-α and metabolism, potentially through its role in regulating UDP-sugars, and reveals a possible novel role for SLC35A2 in lysosomal processing and autophagy. *SLC35A2* expression in renal cell carcinoma is associated with VHL status, which may provide a new route for dysregulation of HIF, altered metabolism, and changes in lysosomal processing and autophagy.","abstract_html":"Background: &lt;br&gt;The hypoxia inducible factor (HIF) family of dimeric transcription factors play a vital role in the cellular response to low oxygen (hypoxia). HIF activation leads to the transcriptional upregulation of a range of genes that are involved in diverse physiological and pathophysiological processes. In malignant tumours increased HIF-α expression, and HIF activation are commonly observed, and are associated with poorer prognosis and disease progression. HIF activity is controlled by a number of different mechanisms, and the identification of molecular regulators of HIF is of intense interest. The Golgi UDP-galactose transporter SLC35A2 was identified by the Ashcroft group as a novel regulator of HIF-α, and CHO cells that had lost SLC35A2 exhibited elevated HIF-α protein in normoxia. It is known that the absence of SLC35A2 in the Golgi leads to wide-ranging glycosylation defects. The underlying mechanisms linking SLC35A2 to HIF-α protein regulation were not known. This thesis aims to further characterise the role effect of SLC35A2 loss on HIF signalling and tumour cell behaviour, in CHO cells and a human cell system, specifically by exploring the hypotheses that these cells may harbour an autophagy defect, or differences in overall UDP-sugar content, in particular O-GlcNAc. Methods and Results: I used parental and Slc35a2 mutant CHO cells, SLC35A2 knockout (KO) and wild type (WT) suspension HeLa (sHeLa) cells, and a panel of cancer cells lines, including 786O, RCC4 renal carcinoma cells which exhibit constitutive HIF activation due to loss of VHL function. Confirming previous work from the Ashcroft group, I showed that Slc35a2 mutant (M6.19) CHO cells exhibit elevated normoxic HIF-1α protein levels and evidence of a glycosylation defect. Expanding on these findings, I found that SLC35A2 loss led to elevated normoxic HIF-α (HIF-1α and HIF-2α) protein and *HIFA* mRNΑ, and elevated expression of HIF-α target genes (*GLUT1*, *VEGF*). Using global RNAseq analysis of SLC35A2 KO and WT sHeLa cells, I found that SLC35A2 loss was associated with highly upregulated expression of a range of genes. Further exploration of the observed glycosylation defect in Slc35a2 mutant CHO cells, showed that SLC35A2 KO sHeLa cells also exhibited altered mobility of GLUT1 protein, which I found was consistent with treatment of WT sHeLa cells with tunicamycin, a protein *N*-glycosylation inhibitor. UDP-sugar analysis by HPLC of Slc35a2 mutant CHO and SLC35A2 KO sHeLa cells indicated increased levels of UDP-GlcNAc compared to their WT counterparts. Furthermore, I found similar patterns of increased UDP-GlcNAc levels in SLC35A2 KO compared to WT sHeLa cells in normoxia and hypoxia, while stable reconstitution of Slc35a2 in Slc35a2 mutant (M6.19) CHO cells reduced levels of UDP-GlcNAc and rescued CMP-sialic acid levels. Consistent with my findings and the involvement of UDP-GlcNAc in glutamine metabolism, I found that Slc35a2 mutant (M6.19) CHO cells were significantly more sensitive to glutamine withdrawal compared to parental (C4.5) CHO cells. Slc35a2 mutant (M6.19) and SLC35A2 KO sHeLa cells exhibited a glycosylation defect of LAMP2A, a protein involved in HIF-1α lysosomal degradation and critical for chaperone mediated autophagy. SLC35A2 mutant (M6.19) CHO cells were significantly more sensitive to the inhibition of proliferation by the inhibitors of autophagy bafilomycin and 3-methyladenine, and showed elevated levels of the autophagy marker LC3B. Finally, from analyses of the TCGA-KIRC database I found *SCL35A2* expression was higher in patients with non-mutant VHL versus mutant VHL in renal cancers. In support of these findings, I showed that basal SLC35A2 protein and mRNA levels were higher in patient-derived 786O renal carcinoma cells reconstituted with wild type VHL (786O-VHL) compared to matched 786-O empty vector control (786O-EV) cells. I also found that high *SLC35A2* expression is associated with poor prognosis in patients with *VHL* non-mutant ccRCC. Conclusion: Taken together, my thesis identifies SLC35A2 as a regulator of HIF-α and metabolism, potentially through its role in regulating UDP-sugars, and reveals a possible novel role for SLC35A2 in lysosomal processing and autophagy. *SLC35A2* expression in renal cell carcinoma is associated with VHL status, which may provide a new route for dysregulation of HIF, altered metabolism, and changes in lysosomal processing and autophagy.","abstract_has_math":false,"creators":["Greef, Basma"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Ashcroft, Margaret"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-09-15","date_published":"2023-09-15","updated_at":"2026-07-22T22:24:13Z","subjects":["Glycosylation","Hypoxia"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/861b198f-fb7e-490d-8b67-27d095b5fda6/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.111202","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Ashcroft, Margaret"]},{"key":"dc:creator","label":"Author","values":["Greef, Basma"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-09-15"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/372337"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Glycosylation","Hypoxia"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/861b198f-fb7e-490d-8b67-27d095b5fda6/download","https://www.rioxx.net/licenses/all-rights-reserved/"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2030-08-09"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.111202"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2008bf28-81c7-4e67-ad7a-8488290f02e0/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Background: <br>The hypoxia inducible factor (HIF) family of dimeric transcription factors play a vital role in the cellular response to low oxygen (hypoxia). HIF activation leads to the transcriptional upregulation of a range of genes that are involved in diverse physiological and pathophysiological processes. In malignant tumours increased HIF-α expression, and HIF activation are commonly observed, and are associated with poorer prognosis and disease progression. HIF activity is controlled by a number of different mechanisms, and the identification of molecular regulators of HIF is of intense interest. The Golgi UDP-galactose transporter SLC35A2 was identified by the Ashcroft group as a novel regulator of HIF-α, and CHO cells that had lost SLC35A2 exhibited elevated HIF-α protein in normoxia. It is known that the absence of SLC35A2 in the Golgi leads to wide-ranging glycosylation defects. The underlying mechanisms linking SLC35A2 to HIF-α protein regulation were not known. This thesis aims to further characterise the role effect of SLC35A2 loss on HIF signalling and tumour cell behaviour, in CHO cells and a human cell system, specifically by exploring the hypotheses that these cells may harbour an autophagy defect, or differences in overall UDP-sugar content, in particular O-GlcNAc. Methods and Results: I used parental and Slc35a2 mutant CHO cells, SLC35A2 knockout (KO) and wild type (WT) suspension HeLa (sHeLa) cells, and a panel of cancer cells lines, including 786O, RCC4 renal carcinoma cells which exhibit constitutive HIF activation due to loss of VHL function. Confirming previous work from the Ashcroft group, I showed that Slc35a2 mutant (M6.19) CHO cells exhibit elevated normoxic HIF-1α protein levels and evidence of a glycosylation defect. Expanding on these findings, I found that SLC35A2 loss led to elevated normoxic HIF-α (HIF-1α and HIF-2α) protein and *HIFA* mRNΑ, and elevated expression of HIF-α target genes (*GLUT1*, *VEGF*). Using global RNAseq analysis of SLC35A2 KO and WT sHeLa cells, I found that SLC35A2 loss was associated with highly upregulated expression of a range of genes. Further exploration of the observed glycosylation defect in Slc35a2 mutant CHO cells, showed that SLC35A2 KO sHeLa cells also exhibited altered mobility of GLUT1 protein, which I found was consistent with treatment of WT sHeLa cells with tunicamycin, a protein *N*-glycosylation inhibitor. UDP-sugar analysis by HPLC of Slc35a2 mutant CHO and SLC35A2 KO sHeLa cells indicated increased levels of UDP-GlcNAc compared to their WT counterparts. Furthermore, I found similar patterns of increased UDP-GlcNAc levels in SLC35A2 KO compared to WT sHeLa cells in normoxia and hypoxia, while stable reconstitution of Slc35a2 in Slc35a2 mutant (M6.19) CHO cells reduced levels of UDP-GlcNAc and rescued CMP-sialic acid levels. Consistent with my findings and the involvement of UDP-GlcNAc in glutamine metabolism, I found that Slc35a2 mutant (M6.19) CHO cells were significantly more sensitive to glutamine withdrawal compared to parental (C4.5) CHO cells. Slc35a2 mutant (M6.19) and SLC35A2 KO sHeLa cells exhibited a glycosylation defect of LAMP2A, a protein involved in HIF-1α lysosomal degradation and critical for chaperone mediated autophagy. SLC35A2 mutant (M6.19) CHO cells were significantly more sensitive to the inhibition of proliferation by the inhibitors of autophagy bafilomycin and 3-methyladenine, and showed elevated levels of the autophagy marker LC3B. Finally, from analyses of the TCGA-KIRC database I found *SCL35A2* expression was higher in patients with non-mutant VHL versus mutant VHL in renal cancers. In support of these findings, I showed that basal SLC35A2 protein and mRNA levels were higher in patient-derived 786O renal carcinoma cells reconstituted with wild type VHL (786O-VHL) compared to matched 786-O empty vector control (786O-EV) cells. I also found that high *SLC35A2* expression is associated with poor prognosis in patients with *VHL* non-mutant ccRCC. Conclusion: Taken together, my thesis identifies SLC35A2 as a regulator of HIF-α and metabolism, potentially through its role in regulating UDP-sugars, and reveals a possible novel role for SLC35A2 in lysosomal processing and autophagy. *SLC35A2* expression in renal cell carcinoma is associated with VHL status, which may provide a new route for dysregulation of HIF, altered metabolism, and changes in lysosomal processing and autophagy."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["87eda9de84448d1f82354d60eee3eb5f","c1c4c1c18a55c6d1c0091c73894cf6c6"]},{"key":"dc:title","label":"Title","values":["Investigating the role of the UDP-galactose transporter SLC35A2 in the regulation of HIF signalling"]}]}],"canonical_facts":{"dc:contributor.advisor":["Ashcroft, Margaret"],"dc:creator":["Greef, Basma"],"dc:date.issued":["2023-09-15"],"dc:description.abstract":["Background: <br>The hypoxia inducible factor (HIF) family of dimeric transcription factors play a vital role in the cellular response to low oxygen (hypoxia). HIF activation leads to the transcriptional upregulation of a range of genes that are involved in diverse physiological and pathophysiological processes. In malignant tumours increased HIF-α expression, and HIF activation are commonly observed, and are associated with poorer prognosis and disease progression. HIF activity is controlled by a number of different mechanisms, and the identification of molecular regulators of HIF is of intense interest. The Golgi UDP-galactose transporter SLC35A2 was identified by the Ashcroft group as a novel regulator of HIF-α, and CHO cells that had lost SLC35A2 exhibited elevated HIF-α protein in normoxia. It is known that the absence of SLC35A2 in the Golgi leads to wide-ranging glycosylation defects. The underlying mechanisms linking SLC35A2 to HIF-α protein regulation were not known. This thesis aims to further characterise the role effect of SLC35A2 loss on HIF signalling and tumour cell behaviour, in CHO cells and a human cell system, specifically by exploring the hypotheses that these cells may harbour an autophagy defect, or differences in overall UDP-sugar content, in particular O-GlcNAc. Methods and Results: I used parental and Slc35a2 mutant CHO cells, SLC35A2 knockout (KO) and wild type (WT) suspension HeLa (sHeLa) cells, and a panel of cancer cells lines, including 786O, RCC4 renal carcinoma cells which exhibit constitutive HIF activation due to loss of VHL function. Confirming previous work from the Ashcroft group, I showed that Slc35a2 mutant (M6.19) CHO cells exhibit elevated normoxic HIF-1α protein levels and evidence of a glycosylation defect. Expanding on these findings, I found that SLC35A2 loss led to elevated normoxic HIF-α (HIF-1α and HIF-2α) protein and *HIFA* mRNΑ, and elevated expression of HIF-α target genes (*GLUT1*, *VEGF*). Using global RNAseq analysis of SLC35A2 KO and WT sHeLa cells, I found that SLC35A2 loss was associated with highly upregulated expression of a range of genes. Further exploration of the observed glycosylation defect in Slc35a2 mutant CHO cells, showed that SLC35A2 KO sHeLa cells also exhibited altered mobility of GLUT1 protein, which I found was consistent with treatment of WT sHeLa cells with tunicamycin, a protein *N*-glycosylation inhibitor. UDP-sugar analysis by HPLC of Slc35a2 mutant CHO and SLC35A2 KO sHeLa cells indicated increased levels of UDP-GlcNAc compared to their WT counterparts. Furthermore, I found similar patterns of increased UDP-GlcNAc levels in SLC35A2 KO compared to WT sHeLa cells in normoxia and hypoxia, while stable reconstitution of Slc35a2 in Slc35a2 mutant (M6.19) CHO cells reduced levels of UDP-GlcNAc and rescued CMP-sialic acid levels. Consistent with my findings and the involvement of UDP-GlcNAc in glutamine metabolism, I found that Slc35a2 mutant (M6.19) CHO cells were significantly more sensitive to glutamine withdrawal compared to parental (C4.5) CHO cells. Slc35a2 mutant (M6.19) and SLC35A2 KO sHeLa cells exhibited a glycosylation defect of LAMP2A, a protein involved in HIF-1α lysosomal degradation and critical for chaperone mediated autophagy. SLC35A2 mutant (M6.19) CHO cells were significantly more sensitive to the inhibition of proliferation by the inhibitors of autophagy bafilomycin and 3-methyladenine, and showed elevated levels of the autophagy marker LC3B. Finally, from analyses of the TCGA-KIRC database I found *SCL35A2* expression was higher in patients with non-mutant VHL versus mutant VHL in renal cancers. In support of these findings, I showed that basal SLC35A2 protein and mRNA levels were higher in patient-derived 786O renal carcinoma cells reconstituted with wild type VHL (786O-VHL) compared to matched 786-O empty vector control (786O-EV) cells. I also found that high *SLC35A2* expression is associated with poor prognosis in patients with *VHL* non-mutant ccRCC. Conclusion: Taken together, my thesis identifies SLC35A2 as a regulator of HIF-α and metabolism, potentially through its role in regulating UDP-sugars, and reveals a possible novel role for SLC35A2 in lysosomal processing and autophagy. *SLC35A2* expression in renal cell carcinoma is associated with VHL status, which may provide a new route for dysregulation of HIF, altered metabolism, and changes in lysosomal processing and autophagy."],"dc:format.checksum.md5":["87eda9de84448d1f82354d60eee3eb5f","c1c4c1c18a55c6d1c0091c73894cf6c6"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.111202"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/2008bf28-81c7-4e67-ad7a-8488290f02e0/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/372337"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/861b198f-fb7e-490d-8b67-27d095b5fda6/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:rights.embargodate":["2030-08-09"],"dc:rights.embargotype":["embargo"],"dc:subject":["Glycosylation","Hypoxia"],"dc:title":["Investigating the role of the UDP-galactose transporter SLC35A2 in the regulation of HIF signalling"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:13Z"}