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Stromal components and micro-RNAs as biomarkers in pancreatic cancer
Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Surgery.ORCID iD: 0000-0002-3777-6887
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background Pancreatic ductal adenocarcinoma (PDAC) patients have the poorest 5-year survival rates of all cancer forms. It is difficult to diagnose at early disease stages, tumour relapse after surgery is common, and current chemotherapies are ineffective. Carbohydrate antigen 19-9 (Ca 19-9), the only clinically implemented PDAC biomarker, is insufficient for diagnostic and screening purposes.

PDAC tumours are characterised by a voluminous stroma that is rich in extracellular matrix (ECM) molecules such as collagens, hyaluronan (HA) and matricellular proteins. These stromal components have been suggested to promote PDAC cell migration, proliferation, evasion of apoptosis and chemotherapy resistance. Those events are mediated via interactions with adhesion receptors, such as integrins and CD44 receptors expressed on cancer cell surfaces.

Micro-RNAs (miRNA) post-transcriptionally regulate gene expression in health and disease. At the time of PDAC diagnosis, miRNA levels are altered both in plasma and tumour tissue. Before PDAC diagnosis, tissue miRNA levels are altered in precursor lesions, raising the possibility that plasma miRNAs might aid in early detection.

In this thesis, it is hypothesised that stromal components and miRNAs can serve as tissue or blood based biomarkers in PDAC. The aims are: (1) to characterise the expression of stromal components and their receptors in normal and cancerous tissue; (2) to find potential stroma-associated tissue and blood-based biomarkers for diagnosis and prognosis estimates; (3) to determine the cellular effects of type IV collagen (Col IV) in PDAC; (4) to determine if plasma miRNAs that are altered in manifest PDAC can be used to diagnose PDAC earlier.

Methods The expression patterns of Col IV, Col IV-binding integrin subunits (α1, α2, β1), Endostatin, Osteopontin (OPN) and Tenascin C (TNC) were analysed in frozen PDAC and normal pancreatic tissue. A tissue microarray (TMA) was constructed using formalin-fixed, paraffin-embedded primary tumours and lymph node metastases. The TMA was used to study the expression levels and associations with survival of the standard CD44 receptor (CD44s), its variant isoform 6 (CD44v6), HA, OPN and Col IV. Circulating levels of HA, Col IV, Endostatin, OPN and TNC were measured in PDAC patients and healthy individuals, and compared with conventional tumour markers (Ca 19-9, CEA, Ca 125 and TPS). The functional roles of Col IV were studied in PDAC cell lines by: (1) growth on different matrices (2) blocking Col IV binding integrin subunits, (3) blocking the Col IV domains 7s, CB3 and NC1, and (4) by down regulation of PDAC cell synthesis of Col IV using siRNA transfection. Plasma miRNAs alterations were screened for in samples from patients with manifest disease, using real-time quantitative PCR (RT-qPCR). To find early miRNA alterations, levels of those miRNAs that were altered at diagnosis were measured in prediagnostic plasma samples.

Results High tissue expression of both the standard CD44 receptor (CD44s) and its variant isoform CD44v6 as well as low expression of stromal OPN were associated with poor survival. In addition, high CD44s and low OPN predicted poor survival independent of established prognostic factors.

Circulating Col IV, Endostatin, OPN, TNC and HA were increased in preoperative samples from PDAC patients. Preoperatively, higher levels of serum-HA and plasma-Endostatin were associated with shorter survival. Postoperatively, higher levels of Col IV, Endostatin and OPN were associated with shorter survival. On the contrary, only one of the conventional tumour markers was associated with survival (Ca 125).

Col IV stimulated PDAC cell proliferation and migration and inhibited apoptosis in vitro, dependent on the collagenous domain (CB3) of Col IV and the Col IV binding integrin subunit β1. Reduced endogenous Col IV synthesis inhibited these effects, suggesting that PDAC cells synthesise Col IV to stimulate tumour-promoting events via a newly discovered autocrine loop.

15 miRNAs were altered in early stage PDAC patients and the combination of these markers outperformed Ca 19-9 in discriminating patients from healthy individuals. However, none of the miRNAs were altered in prediagnostic samples, suggesting that plasma miRNA alterations appear late in the disease course.

Conclusions Up regulated stromal components in PDAC tumours are detectable in blood samples and are potential diagnostic and prognostic biomarkers in PDAC. High circulating levels of Col IV, Endostatin, OPN and HA predict poor survival, as well as high expression of CD44s and CD44v6 and low expression of OPN in tumour tissue. PDAC cells synthesise Col IV, which forms BM-like structures close to cancer cells and promote tumour progression in vitro via an autocrine loop. Several plasma-miRNAs are altered in PDAC, but are not useful for early discovery. 

Place, publisher, year, edition, pages
Umeå: Umeå University , 2016. , 64 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1833
Keyword [en]
Pancreatic cancer, stroma, basement membrane, tumour markers, type IV collagen, endostatin, osteopontin, tenascin c, hyaluronan, CD44, micro-RNA
National Category
Cancer and Oncology Surgery
Research subject
Surgery
Identifiers
URN: urn:nbn:se:umu:diva-128000ISBN: 978-91-7601-454-7OAI: oai:DiVA.org:umu-128000DiVA: diva2:1048471
Public defence
2016-12-16, Hörsal B, målpunkt A, 9 trappor, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilThe Kempe FoundationsSwedish Society of MedicineSwedish Cancer Society
Available from: 2016-11-25 Created: 2016-11-21 Last updated: 2016-11-23Bibliographically approved
List of papers
1. Type IV collagen stimulates pancreatic cancer cell proliferation, migration, and inhibits apoptosis through an autocrine loop
Open this publication in new window or tab >>Type IV collagen stimulates pancreatic cancer cell proliferation, migration, and inhibits apoptosis through an autocrine loop
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2013 (English)In: BMC Cancer, ISSN 1471-2407, E-ISSN 1471-2407, Vol. 13, 154Article in journal (Refereed) Published
Abstract [en]

Background: Pancreatic cancer shows a highly aggressive and infiltrative growth pattern and is characterized by an abundant tumor stroma known to interact with the cancer cells, and to influence tumor growth and drug resistance. Cancer cells actively take part in the production of extracellular matrix proteins, which then become deposited into the tumor stroma. Type IV collagen, an important component of the basement membrane, is highly expressed by pancreatic cancer cells both in vivo and in vitro. In this study, the cellular effects of type IV collagen produced by the cancer cells were characterized.

Methods: The expression of type IV collagen and its integrin receptors were examined in vivo in human pancreatic cancer tissue. The cellular effects of type IV collagen were studied in pancreatic cancer cell lines by reducing type IV collagen expression through RNA interference and by functional receptor blocking of integrins and their binding-sites on the type IV collagen molecule.

Results: We show that type IV collagen is expressed close to the cancer cells in vivo, forming basement membrane like structures on the cancer cell surface that colocalize with the integrin receptors. Furthermore, the interaction between type IV collagen produced by the cancer cell, and integrins on the surface of the cancer cells, are important for continuous cancer cell growth, maintenance of a migratory phenotype, and for avoiding apoptosis.

Conclusion: We show that type IV collagen provides essential cell survival signals to the pancreatic cancer cells through an autocrine loop.

Keyword
Type IV collagen, Pancreatic cancer, Basement membrane, Integrin receptors, Autocrine loop
National Category
Surgery Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-71100 (URN)10.1186/1471-2407-13-154 (DOI)000317397700001 ()
Available from: 2013-06-12 Created: 2013-05-20 Last updated: 2016-11-23Bibliographically approved
2. Combining conventional and stroma-derived tumour markers in pancreatic ductal adenocarcinoma
Open this publication in new window or tab >>Combining conventional and stroma-derived tumour markers in pancreatic ductal adenocarcinoma
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2015 (English)In: Cancer Biomarkers, ISSN 1574-0153, Vol. 15, no 1, 1-10 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: A lack of disease-specific symptoms and good tumour markers makes early detection and diagnosis of pancreatic ductal adenocarcinoma (PDAC) challenging. OBJECTIVE: To analyse the tissue expression and circulating levels of four stroma-derived substances (type IV collagen, endostatin/type XVIII collagen, osteopontin and tenascin C) and four conventional tumour markers (CA 19-9, TPS, CEA and Ca 125) in a PDAC cohort.

METHODS: Tissue expression of markers in normal pancreas and PDAC tissue was analysed with immunofluorescence. Plasma concentrations of markers were measured before and after surgery. Patients with non-malignant disorders served as controls.

RESULTS: The conventional and stromal substances were expressed in the cancer cell compartment and the stroma, respectively. Although most patients had increased levels of many markers before surgery, 2/12 (17%) of patients had normal levels of Ca 19-9 at this stage. High preoperative endostatin/type XVIII collagen, and postoperative type IV collagen was associated with short survival. Neither the pre-nor postoperative levels of TPS, Ca 125 or CA 19-9 were associated to survival.

CONCLUSIONS: PDAC is characterized by an abundant stroma. These initial observations indicate that the stroma can be a source of PDAC tumour markers that are found in different compartments of the cancer, thus reflecting different aspects of tumour biology.

Place, publisher, year, edition, pages
IOS Press, 2015
Keyword
Pancreatic ductal adenocarcinoma (PDAC), tumour markers, stroma, type IV collagen, type XVIII llagen, endostatin, osteopontin, tenascin C, TPS, Ca 125, Ca 19-9, CEA
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-97877 (URN)10.3233/CBM-140430 (DOI)000346079800001 ()
Available from: 2015-01-16 Created: 2015-01-08 Last updated: 2016-11-23Bibliographically approved
3. CD44 receptors and stromal CD44 ligands as prognostic markers in pancreatic ductal adenocarcinoma
Open this publication in new window or tab >>CD44 receptors and stromal CD44 ligands as prognostic markers in pancreatic ductal adenocarcinoma
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-127997 (URN)
Available from: 2016-11-21 Created: 2016-11-21 Last updated: 2016-11-23
4. Plasma micro-RNA alterations appear late in pancreatic cancer
Open this publication in new window or tab >>Plasma micro-RNA alterations appear late in pancreatic cancer
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-127998 (URN)
Available from: 2016-11-21 Created: 2016-11-21 Last updated: 2016-11-23

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