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Role of Primary Cilia in Ovarian Cancer

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, tagged AURA, aurora kinase, CHFR, cilia, Gli factors, hedgehog, ovarian cancer, pdgf, Transcriptional Activator-Coactivator Interactions on January 15, 2013| 5 Comments »

Role of Primary Cilia in Ovarian Cancer

Curator: Aashir Awan, PhD

Word Cloud By Danielle Smolyar

Each year, over 20 000 women in the US are diagnosed with ovarian cancer while there are nearly 15 000 deaths (http://www.cdc.gov/uscs). The 5-year (post-diagnosis) mortality rate is less than 46% partly due to the fact that early detection still remains difficult (Jemal et al., 2010). So, it is not surprising to see biomedical research putting a much needed focus on this deadly killer.

As such, a joint collaboration between Dr. Søren T Christensen’s lab (University of Copenhagen, Denmark) and the Laboratory of Reproductive Biology (LRB, RigsHospital, Copenhagen) decided to investigate into the specific molecular mechanisms behind ovarian cancer. This resulted in the recent publication linking aberrant hedgehog/PDGF signaling and ovarian cancer (Egeberg et al., 2012). Specifically, the paper shows that there are specific defects associated with the primary cilium of these cancer cell lines which then lead to a perturbation of two critical signaling systems that were examined in the paper. The starting materials included ovarian cancer lines OVCAR3 and SK-OV3. And, the control WT cells are ovarian surface epithelium (OSE which are thought to be the cells of origin for ovarian cancer) cells that were obtained at LRB where young girls that are diagnosed with cancer can deposit and cryopreserve their ovaries for future implantation after being deemed cancer-free.

Biomarkers evaluated in the study included

1. Gli proteins – glioma transcription factors specific to hedgehog signaling (regulating cell proliferation/differentiation). In the off stage, they are thought to act as repressors while in the on state, the proteins are modified at the primary cilium to become activators (Satir el al., 2010).

2. CHFR (checkpoint with forkhead-associated and ring finger domains) – a tumor suppressor gene with multiple functions in checkpoints during mitosis (Kang et al., 2002)

3. AURA (Aurora Kinase A) mark centrosome maturation, mitotic entry an spindle assembly (Fu et al., 2007).

4. PDGF (platelet derived growth factor) is a signaling pathway involved in cell growth/survival, differentiation, migration and wound healing (Schneider et al., 2010).

The paper begins with characterizing the epithelial nature of OSE to confirm the validity of the primary OSE cell cultures. Of note is the fact that OSE lack E-cadherins present in other epithelial cells while expressing N-cadherin and vimentin which together suggest OSE have a more mesenchymal characteristic in comparison to other epithelial tissues. Meawhile, OVCAR3 was negative for vimentin while both cancer cell lines expressed N-cadherin and E-cadherin (more so) indicating these cancer cell lines retained more of a classic epithelial characteristic in comparison to WT OSE which might imply lack of plasticity (Wong et al., 1999).

Next, ciliogenesis (ability to form a primary cilium) was evaluated in the cancer cell lines vs WT OSE. Under serum starvation (used to induce cilia formation), far fewer cancer cells are able to form a primary cilium. Since primary cilium are formed in mitotically inactive cells (usually at G0), it was speculated that the lack of cilia in the cancer lines implied greater proliferation rates. However, Ki67, phosphorylated retinoblastoma protein (p-RB) and PCNA (cell proliferation markers) stainings showed that under serum starvation conditions, the cancer cells still have the ability to enter growth arrest.

Fig3a

So, there had be another explanation accounting for the difference between WT OSE and the cancer cell lines such as a interruption of signal between the primary cilium and the growth arrest apparatus. Thus, the hedgehog signaling was then examined. Full-length Gli2 acts as a repressor while cleavage at the primary cilia results in the activator form. WT OSE had very little full-length Gli2 whereas the cleaved repressor form is present. This indicates that the hedgehog pathway is mostly inactive in WT OSE. On the other hand, the two cancer lines had higher levels of the full length activator form and little of the cleaved repressor form. Serum starvation decreased this activation of the hedgehog signal as expected (due to the presence of the primary cilium cleaving the Gli2) but not down to the same levels as in WT OSE. This was confirmed by RT-PCR results which in summary stated that there is a higher basal level of hedgehog signaling in the cancer lines vs WT OSE.

PDGFa had previously been reported to be upregulated during serum starvation and consequently is seen accumulating at the primary cilium (Schneider et al., 2005). While this phenomenon is observed In WT OSE, not only are there lower levels of PDGF overall, but there is no accumulation of this protein upon serum starvation. Therefore, PDGF signaling is perturbed in the cancer cell lines.

Next, the expression of AURA was examined. This kinase is responsible for ciliary disassembly which is a necessary step before entering mitosis. The cancer OSE cells were shown to have higher AURA levels as compared to WT OSE. Given that these cancer cell lines have fewer primary cilia, one can deduce that the higher AURA protein levels suppress ciliogenesis while in WT OSE, they have a role in ciliary disassembly. This was further confirmed by the fact that knockdown of AURA in the cancer cell lines resulted in increased ciliogenesis.

Then, the paper looks upstream of AURA at CHFR which is responsible for the ubiquitination/ proteosomal degradation of AURA. During serum starvation (i.e. primary cilia is formed), CHFR is found at the base of the cilium in WT OSE and this localization disappears during mitosis. In the transformed hTERT-RPE1 cells, CHFR is found both in growth-arrested as well as cells in mitosis. Therefore, this result hints at a AURA/CHFR interaction at the centrosome regulating ciliary dynamics.

Thus, an axis linking the primary cilium to different signal transduction systems and eventually leading to cancer is shown below:

Cilia defect Hedgehog/PDGF uncontrolled proliferation cancer

The cilia defect is likely due to a mutation in the AURA/CHFR axis or even further upstream. While this mechanistic explanation summarizes the findings of this paper,its logic can be applied to other cancer types also (Veland et al., 2009).

REFERENCES

Egeberg DL, Lethan M, Manguso R, Schneider L, Awan A, Jørgensen TS, Byskov AG, Pedersen LB, Christensen ST (2012) Primary cilia and aberrant cell signaling in epithelial ovarian cancer. Cilia 1:1-15.

Fu J, Bian M, Jiang Q, Zhang C (2007) Roles of Aurora kinases in mitosis and tumorigenesis. Mol Cancer Res 5:1–10.

Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300.

Kang D, Chen J, Wong J, Fang G (2002) The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition. J Cell Biol 156:249–259.

Satir P, Pedersen LB, Christensen ST (2010) The primary cilium at a glance. J Cell Sci. 123:499-503.

Schneider L, Clement CA, Teilmann SC, Pazour GJ, Hoffmann EK, Satir P, Christensen ST (2005) PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. Curr Biol 15:1861-1866.

Schneider L, Cammer M, Lehman J, Nielsen SK, Guerra CF, Veland IR, Stock C, Hoffmann EK, Yoder BK, Schwab A, Satir P, Christensen ST (2010) Directional cell migration and chemotaxis in wound healing response to PDGF-AA are coordinated by the primary cilium in fibroblasts. Cell Physiol Biochem 25:279-292.

Veland IR, Awan A, Pedersen LB, Yoder BK, Christensen ST (2009) Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol 111: 39-53.

Wong AS, Maines-Bandiera SL, Rosen B, Wheelock MJ, Johnson KR, Leung PC, Roskelley CD, Auersperg N (1999) Constitutive and conditional cadherin expression in cultured human ovarian surface epithelium: influence of family history of ovariancancer. Int J Cancer 81:180–188.