The significance of PAX6 in lung cancer and sarcoma
Transcription factor PAX6 has a decisive role in the development of the CNS, eye, pancreas and olfactory epithelium, and the pattern of PAX6 normal expression is restricted to those tissues. PAX6 is detected in various cancer cell lines, but very little is known about its expression in tumors, possible cancer-relevant functions and prognostic value. PAX6 is generally believed to be a tumor suppressor, based mainly on studies in glioblastoma. PAX6 has been detected in tumors of eye and pancreas, but the prognostic significance remains unclear. Soft tissue sarcomas (STS) are a relatively uncommon but deadly group of tumors with poor prognosis and few treatment options. We had previously detected PAX6 in STS - the first tumor group with no developmental links to the normal sites of PAX6 expression. PAX6 appeared to confer a universal negative prognostic impact on DSS, irrespectively of clinical and pathological subgroups analyzed. PAX6 expression correlated with TGF-beta, and reciprocal upregulation may explain their synergistic negative effect on DSS. The financing from Helse Nord allowed postdoc fellow Yury Kiselev to gain experience in various methods and their results’ interpretations, including: establishment of stable knockdowns of putative oncogenes in human and mammalian cancer cell lines with the use of lentiviridae, maintenance and RNAi interference in human soft tissue sarcoma cell lines, cell migration analysis with the use of silicon microinserts, proliferation analysis with the use of MTT method, apoptosis analysis with the use of Caspase Glo 3/7 method, work with mRNA expression databases, analysis of gene expression profiles, immunohistochemical staining of rhabdomyosarcoma samples for PAX6. In regards to soft tissue sarcomas, we received data indicating that PAX6 upregulates proliferation of several sarcoma cell lines, inhibits apoptosis (activation of caspase 3/7) and stimulates cell migration in the wound healing assay. However, for most of the analyses there was no statistical significance, possibly due to high variability of RNAi transfection efficiency. Another challenge was the heterogeneity of the STS tumor group, and it was not possible to cover all the range of subgroups of STS in our TMA data set with corresponding cell lines, as the latter are very rare. Hence, the data obtained on PAX6 prognostic effect in soft tissue sarcomas will need to be published alone, with planned submission in March 2016. Lung cancer is the major reason of death from cancer in Norway. Long-term survival is low, and even modern high-cost treatments have only limited efficiency. We have obtained promising data on the prognostic significance of PAX6 for non-small cell lung cancer, yet statistical significance of those is limited only to node-positive subgroup of patients. An addition, there have been found PAX6-specific biological effects in lung cancer cells, such as modulation of cell adhesion and cell migration, which are in line with observed prognostic effect. The manuscript on that has been prepared and submitted to PLoS ONE in January 2016. In addition, financing from Helse Nord has been used to obtain essential data for completion of the manuscript on the regulatory role of PAX6 towards famous tumor suppressor BRCA2 in prostate cancer cells. The manuscript is going to be submitted in February 2016. We have also attempted to study effect of PAX6 in breast and colon cancer, but our TMA collection didn't show any significant expression of PAX6 in those tumors.
The data obtained in frames of this project indicates that PAX6 has prognostic significance for patients with soft tissue sarcomas and non-small cell lung cancer. In addition, we have shed more light on biological role of PAX6 in cancer. This together may lead to better understanding of cancer ethiology and pathogenesis, as well as to development of targeted therapies.
The significance of PAX6 in lung cancer and sarcoma
Prosjektet har gått som planlagt.
Man har sammenlignet PAX6 sin rolle i utvikling av lungekeft og sarkom. Prosjektet er basert på funksjonelle studier av kreftcellelinjer in vitro og sammenholdt med PAX6 sin rolle som prognosisk markør i store pasientkohorter med hhv lungekreft og sarkom. Det er framkommet ny og interessant kunnskap som vil bli publisert i form av tre artikler i nær framtid.
Lung cancer are endodermal derived tumors and is the leading cause of cancer death in the western world (1). The high mortality rate is related to the low 5-year survival rate (6-15%), which in turn is related to the lack of adequate screening and early detection measures (2). There are two main categories of lung cancer: NSCLC (80%) and small cell lung cancer (SCLC; 20%). Even among patients treated for stage I-III NSCLC and considered postoperatively tumour-free, about 65% will relapse within two years after surgery and subsequently die of metastatic spread (3, 4).
Soft tissue sarcomas (STS) are mesodermal derived tumors making up about 0.5% of the annual cancer incidence in the US (5). The STS group consists of more than 50 histological entities. Because of the low incidence and similar ancestry of these tumors it is convenient to group them together when conducting studies (6). A proposed way to group these tumors are Ewing family tumors, gastrointestinal stromal tumors (GISTs) and (non-GIST) STSs, with the latter group consisting of the remaining tumors (7). Despite improvements in therapy over the last decades the disease-specific survival and progression free survival of sarcoma patients are still poor. The main treatment is resection with wide margins, while radiotherapy is often used in high-grade tumors with both marginal and wide resection margins (8, 9). Several adjuvant chemotherapy-regimes are used in the treatment of sarcomas, but with the exception of childhood rhabdomyosarcomas, Ewing family tumors and GISTs, studies are inconclusive on the effects of these agents (8, 10).
The role of PAX6 as predictor for treatment response and survival in non-small cell lung cancer (NSCLC) and soft tissue sarcoma (STS) will be explored in this study. PAX6 belongs to the paired family of transcription factors and have dissimilar impact in endodermal derived cancers and mesodermal derived cancers. Our tissue micro arrays of cohorts of patients with these two main cancer classes, show that the expression of PAX6 is significant positive correlated with disease specific survival in lung cancer patients, but significant negative correlated with survival in sarcoma patients. We have recently shown that knockdown of PAX6 in an adenocarcinoma cell line (endodermal derived) leads to increased proliferation, while transfection of PAX6 into 3T3 fibroblasts (mesodermal derived) activates their proliferation, adhesion and migration. In this project we want to increase the understanding of the role of PAX6 in cancers of different embryonic germ layers by employing basic cell biological and molecular techniques on isolated cancer cells and stromal cells from resected tumors from patients and commercial cell lines. Clinical significance of any laboratory results will be validated in large cohorts of patients with NSCLC and STS. PAX6 influences cancer progression, metastasis and treatment response and therefore has a potential to be an important prognostic marker and may be useful for choosing the optimal treatment options.
Clinical significance and biological effects of PAX6 in non-small cell lung cancer and soft tissue sarcomas
Based on the previously established pro-survival effect of PAX6 in lung cancer patients, we went into elucidating the biological and molecular events explaining this effect. We have found that PAX6 inhibits tumour cell migration, though having little effect on proliferation and apoptosis. PAX6 regulates genes involved in building new blood vessels.
Our aim in 2013 was to find out what kind of biological and molecular events/phenomena may explain clinical significance of PAX6, namely that it confers positive prognostic impact on survival of patients with non-small cell lung cancer (significant in the subgroup of patients with local lymph node spread). We used a special system xCELLigence which allowed us to track in real time what happens to the major biological activities of cells (proliferation, migration) after knock-down of PAX6. We found out that PAX6 has variable effect on proliferation of various NSCLC cell lines, but a strong reproducible inhibitory effect on cell migration along the serum gradient. This finding was further reproduced with the use of an image-analysis machine Incucyte, which showed that cells with PAX6 knockdown migrated better than control cells. We also found that PAX6 regulates apoptosis in several NSCLC cell lines.
We found that PAX6 has a regulatory effect on transcription of various cancer-relevant genes. A prominent number of those related to angiogenesis - a process of building new blood vessels to feed the growing tumour. The difficulty with this transcription data was the high fluctuation of observed changes in repeated experiments, which demanded 5-7 replicates to be performed.
We have started to write a paper on PAX6 in non-small cell lung cancer, which is expected to be submitted before spring 2014. We have started a collaboration with Hamburg Eppendorf Hospital to create stable knockdown cell lines to be used in in vivo models to verify PAX6 effect on tumour growth in vivo. On the soft tissue sarcomas we have strong prognostic data and some molecular indications that PAX6 collaborates with TGFb in conferring more aggressive features to the tumour. However, due to the absence of good quality cell lines we couldnt perform proper investigations of biological effects of PAX6 in sarcomas. We are going to start a collaboration with a foreign centre of excellence in soft tissue sarcomas to perform such experiments. For the moment, an abstract on PAX6 effect in soft tissue sarcomas has been submitted and accepted for American Association of Cancer Research annual conference in 2014.
The significance of PAX6 in lung cancer and sarcoma
We are first to discover expression of PAX6 in biopsies from NSCLC and soft tissue sarcoma patients. PAX6 promotes survival of NSCLC patients with node+ status. We found a strong negative impact of PAX6 on survival in STS.
We found that PAX6 regulates tissue factor - relevant for cancer progression and spontaneous thrombosis in cancer patients.
Throughout the year 2012 the project involved the following main activities: laboratory installation, introducing new research methods, acquiring molecular and clinical data.
A) Laboratory installation and methods. We have optimized manual and automated immunohistochemical staining procedures for PAX6 detection in tissue sections. Two methods for studying cell proliferation and migration have been implemented: xCELLigence and Incucyte, as well as apoptosis detection method based on caspase3/7 fluorescence induction. We have created a collection of human cell lines originated from lung cancer and other malignancies.
B) Acquiring new molecular and clinical data was performed by means of studying PAX6 effects on gene expression profile, protein expression profile, biological processes (cell proliferation, migration) and clinical outcomes (survival, invasion, metastasis, etc).
We are the first group to discover expression of PAX6 in clinical material from NSCLC patients. Based on our initial finding that PAX6 promotes survival of NSCLC patients who have a regional lymph nodes’ involvement, we looked into biological effects of PAX6 in NSCLC cell lines. We discovered strong inhibitory effect of PAX6 on cell migration, which is at least partial explanation of our clinical data. The paper is under preparation and will be submitted in spring 2013.
In clinical material of soft tissue sarcomas we are first to discover expression of PAX6. We found a strong negative impact of PAX6 on survival, in fact this effect knocked out all other identified prognostic factors in a multifactorial analysis. We are now purchasing STS cell lines to search for biological mechanisms of this prognostic effect. The paper is going to be submitted in the summer 2013.
We have finalised laboratory work on regulation of tissue factor by PAX6, which is our novel finding and has implications for cancer progression and spontaneous thrombosis in cancer patients. The paper will be written and submitted within spring 2013.
In a series of experiments we discovered a previously unknown regulation of PAX6 expression by hypoxia in a variety of cancer cell lines, including NSCLC. We have also found that PAX6 regulates apoptosis through activation/inhibition of caspases 3 and 7. Also, we observed significant effects of PAX6 on cancer cell proliferation and migration, including NSCLC cells. We have utilized a panel of primers against genes relevant for normal development and cancer to evaluate possible downstream mechanisms of PAX6 action in human tumours. Based on comparing gene-specific knockdown versus control sample, we have identified the following genes to be regulated by PAX6 in two NSCLC cell lines (derived from adeno- and squamous-cell carcinomas): TGFB1, VEGFA, VEGFD, VEGFR2, VEGFR3, CTGF, PTEN, TNFa. We are currently testing whether the proteins coded by these genes are also expressed differently depending on the presence of PAX6. These are going to be subjects of studies in 2013.
Stromal expression of MiR-21 predicts biochemical failure in prostate cancer patients with Gleason score 6.
PLoS One 2014;9(11):e113039. Epub 2014 nov 17
Monocarboxylate transporters 1-4 in NSCLC: MCT1 is an independent prognostic marker for survival.
PLoS One 2014;9(9):e105038. Epub 2014 sep 16
Pax6 regulates the expression of Dkk3 in murine and human cell lines, and altered responses to Wnt signaling are shown in FlpIn-3T3 cells stably expressing either the Pax6 or the Pax6(5a) isoform.
PLoS One 2014;9(7):e102559. Epub 2014 jul 16
Discovery and verification of BRCA2 as a new target gene for PAX6.
to be submitted February 2016
PAX6 improves survival in node-positive non-small cell lung cancer patients.
Submitted to PLoS ONE, January 2016