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European Biopharmaceutical Review

Under Investigation

Stem cells are unique from all other bodily cells thanks to three traits: firstly, they are capable of dividing and renewing themselves for long periods, unlike muscle or nerve cells; secondly, they are unspecialised; and finally, they can differentiate into specialised cells (1).

Scientists are trying to understand two fundamental properties in relation to their long-term self-renewal:

  • Why can embryonic stem cells proliferate indefinitely in the laboratory without differentiating, when most adult stem cells cannot?
  • What are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?
Study Interests

Adult stem cells – also referred to as somatic stem cells – have been identified in many organs and tissues, including the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium and testis. They are believed to reside in specific areas of each tissue in the so-called ‘stem cell niche’ and can remain quiescent for long periods of time until they are activated to generate more cells to maintain normal tissues, or cells lost due to disease or injury.

Researchers are hoping to identify how undifferentiated stem cells can become differentiated cells in developing tissues and organs, as some of the most serious medical conditions – such as cancer and birth defects – are due to abnormal cell division and impaired differentiation. Particularly important are transcription factors: proteins that directly or indirectly interact with DNA, thereby switching genes on or off during embryo development. For instance, Oct-4 and Nanog are two crucial transcription factors associated with maintaining pluripotent stem cells in an undifferentiated state, capable of self-renewal.

Stem cell research can be applied to a number of different areas:

Cancer Research

Sex determining region Y-box 2 (SOX2) is another important transcription factor that is critical to the formation of many different tissues and organs during embryonic development. Numerous studies have been carried out examining the importance of the SOX2 gene and its relationship to various cancers such as lung, breast, oesophageal and brain tumours.

Belgian scientists found that in mice, SOX2 was the most upregulated transcription factor expressed in stem cells in skin squamous-cell carcinoma (SCC) or tumours (2). It plays a key role in the initiation and progression of skin cancer; in normal skin, SOX2 is not expressed, but begins expression in early stages of tumour formation. When SOX2 is deleted in mice upon inducing carcinogenesis, a decrease in tumour formation is noted. This mechanism is different from other SCC due to genetic alterations.

Non-small cell lung cancer (NSCLC) is a result of genetic alterations containing stem-like cells that are responsible for lung tumour initiation, maintenance, relapse and metastasis (3). In SCC, a subtype of NSCLC, investigators have found that oncogene, protein kinase C or PRKC1 located at chromosome 3q26 and SOX2 are coamplified and coordinately over-expressed in lung squamous cell carcinoma (LSCC) (3). PRKC1 is required for the growth of lung cancer cells, while tumour protein kinase C iota (PKCι) drives lung squamous carcinoma cell invasion and transformed growth in vitro and in vivo. PKCι expression is predictive of poor clinical outcome.

Looking into SOX2
Researchers found that PKCι phosphorylation of SOX2 led to immediate upregulation of hedgehog acyltransferase (HHAT). This is required as the rate-limiting step for activating the signalling pathway. They found that SOX2-mediated HHAT upregulation drives the hedgehog pathway activation towards the maintenance of stem-like cells in lung SCC. Three conclusions resulted from their study:

  • PKCι and SOX2 are genetically linked, being co-amplified on chromosome 3q26 in lung SCC
  • PKCι and SOX2 are biochemically linked, as SOX2 serves as a substrate for PKC phosphorylation
  • PKCι and SOX2 are functionally linked, as they maintain the stem-like properties of these lung cancer cells to drive tumourigenesis (4)
The major gene associated with skin melanoma is CDKN2A/p16, cyclin dependent kinase inhibitor 2A, which is located on chromosome 9p21 (5). Hedgehog-GLI (HH-GLI) signalling is required for melanoma growth, and survival and expansion of melanomainitiating cells. It also regulates genes expressed in embryonic stem cells – including SOX2 – with transcription factors GLI1 and GLI2. While SOX2 is over-expressed in melanoma stem cells and knockdown, silencing and depletion of SOX2 leads to a decrease in self-renewal in melanoma spheres, inhibits cell growth, induces apoptosis in melanoma cells and significantly decreases tumour-initiating capability of high aldehyde dehydrogenase activity. This suggests that SOX2 is required for tumour initiation and for continuous tumour growth (6).

SOX2 was over-expressed in three examples of skin SCC, melanoma and LSCC, and played a critical role in self-renewal, initiation and progression of cancer growth. The absence of SOX2 demonstrated that cancer cell initiation and growth can be decreased and perhaps eliminated; however, deleting or silencing the gene is not feasible, because it is also important for normal cell growth. Finding the SOX2 on-off switch is crucial to prevent cancer, but that is easier said than done.

Major Breakthroughs

Since the completion of the first human genome sequence in 2003, scientists have been trying to figure out which regions of the genome play an important part in the development of diseases. They have hypothesised that the regions that turn genes on and off are key.

Recently, researchers from the University of Toronto, Canada, discovered the relationship between the SOX2 gene – critical for early development – and a region located somewhere else on the genome that effectively regulates its activity (7). Professor Jennifer Mitchell, lead investigator, studied how the SOX2 gene is turned on in mice by finding the region of the genome that is needed to turn on the gene in embryonic cells.

“Like the gene itself, this region of the genome enables these stem cells to maintain their ability to become any type of cell, a property known as pluripotency. We named the region of the genome that we discovered the SOX2 control region, or SCR,” said Mitchell. “The parts of the human genome linked to complex diseases, such as heart disease, cancer and neurological disorders, can often be far away from the genes they regulate, so it can be difficult to figure out which gene is being affected and ultimately causing the disease.”

“We then focused on the region we have since named the SCR, as my work had shown that it can contact the SOX2 gene from its location 100,000 base pairs away,” said Harry Zhou, lead author and former graduate student in Mitchell’s laboratory. “To contact the gene, the DNA makes a loop (chromatin loops) that brings the SCR close to the gene itself only in embryonic stem cells. Once we had a good idea that this region could be acting on the SOX2 gene, we removed the region from the genome and monitored the effect on SOX2” (7).

Positive Outcomes

Results revealed that two gene-proximal enhancers – SOX2 regulatory region 1 (SRR1) and regulatory region 2 (SRR2) – showed activity, but SRR2 was more crucial, as deleting SRR1 had no effect on pluripotency. The assay also discovered three novel enhancers – SRR18, SRR107 and SRR111 – that form chromatin loops by forming a chromatin complex with the SOX2 promoter in embryonic stem cells. Only the distal cluster containing SRR107 and SRR111, located greater than 100kb downstream from SOX2, is required for cis-regulation of SOX2 in embryonic stem cells (8).

“Just as deletion of the SOX2 gene causes the very early embryo to die, it is likely that an abnormality in the regulatory region would also cause early embryonic death before any of the organs have even formed,” said Mitchell. “It is possible that the formation of the loop needed to make contact with the SOX2 gene is an important final step in the process by which researchers practising regenerative medicine can generate pluripotent cells from adult cells” (9).

Drug Testing and Development

Human cells or tissues are used in standard new drugs testing, and cancer cell lines have been utilised to test anti-tumour medications. However, the standard method is not always reliable, because there can be varied responses due to genetic variation from donor to donor, and human cells are in short supply.

About two years ago, pharma companies started incorporating human stem and differentiated cells from induced pluripotent stem cells (iPSCs) when testing new drugs. Researchers from Edinburgh, UK, were able to generate liver cells that were of the same quality as cells from human liver tissue to assess drug safety. They developed a technique to overcome the genetic variability in donor tissue and were able to make uniform liver cells in the lab (10).

In addition, GE Healthcare and Stephen Munger, a stem cell biologist, demonstrated that their heart cells were able to identify which compounds were toxic out of a set of compounds in a blind trial (11). Therefore, using iPSCs gives researchers the ability to perform drug testing on a wide range of cell types.

Organ-On-Chip
Currently, scientists at the Wyss Institute are developing miniature organs using stem cells called ‘organ-on-chip’ for drug testing and development (12). The goal behind this is to eliminate animal model testing since it is not representative of the human system, and is expensive and time-consuming to perform. Eventually, they hope to develop ten organs on a chip, and link them together to mimic the human body in safety and efficacy testing.

Unfortunately, the creation and scaleup of quality stem cells is difficult to achieve. To screen for drugs effectively, scientists must be able to create identical conditions by precisely controlling cell differentiation. However, the signalling mechanism for controlling differentiation is not well-known in some cell types and tissues, making it harder to mimic identical conditions. More work needs to be done before researchers can achieve this.

For Transplantation


Investigators are trying to grow adult stem cells in cell culture and manipulate them to generate specific cell types to treat injuries and diseases. Potential approaches include regenerating bone using cells derived from bone marrow stroma, developing insulin-producing cells for type 1 diabetes, and repairing damaged heart muscle following a heart attack with regenerated cardiac muscle cells.

However, in each tissue, there are a very limited number of stem cells, and once removed from the body their capacity to divide is limited, thus making it difficult to generate large quantities. If scientists can reliably direct embryonic stem cells to differentiate into specific cell types, they may be able to use these to target certain diseases in the future. Conditions that could be treated include diabetes, traumatic spinal cord injury, Duchenne muscular dystrophy, heart disease, and vision and hearing loss.

If scientists can use the patient’s own embryonic cells or iPSCs, and differentiate them into specific cell types before reintroducing them into the body – much like immunotherapy – there would be a lower chance of rejection by the immune system. This would be a significant advantage, as the only recourse to rejection is the continuous administration of immunosuppressive drugs, which can have deleterious side-effects.

Promising Future

In 2006, Shinya Yamanaka and colleagues made a breakthrough discovery by showing that skin fibroblasts can be reprogrammed into embryonic stem cell-like cells. These induced iPSCs hold tremendous promise for regenerative medicine and drug discovery. However, being able to control cell proliferation and differentiation predictably and safely, will require more basic research into the molecular and genetic signals that regulate cell division and specialisation.

Transcription factors – such as Oct4, Nanog, SOX2, KLF4 and c-Myc – are crucial for the regulation of embryonic development and cell differentiation. These same factors also play important roles in cancer biology. As noted above, SOX2 over-expression is involved in selfrenewal, initiation and cancer progression, and manipulation of SOX2 significantly influences the fate of cancer cells.

It has also been demonstrated that the SOX2 regulatory region – particularly the SRR2 – determines the fate of cells, as deletion of SRR1 does not affect pluripotency. An abnormality in the regulatory region not only leads to embryonic death, but may contribute to the initiation of cancer cells and the binding of the chromatin loop that starts and continues the process of development. Finding methodologies to turn off this cis-regulation at the appropriate time and duration – without affecting normal cells – could be an interesting next challenge.

Techniques used to develop iPSCs have given us a greater understanding of how to control some aspects of cell proliferation and differentiation, but further research is also needed in order to be able to manipulate cells predictably and safely. Unanticipated mishaps may lead to cells of the wrong type that may cause rejection in cell transplantation or, even worse, cancer.

Currently, viruses are used to introduce the reprogramming factors in adult cells. However, in animal studies, the virus used to introduce stem cell factors sometimes causes cancers, and researchers are currently investigating non-viral delivery strategies (1).

We have observed that SOX2 is genetically, biochemically and functionally linked to PKCι in driving tumorigenesis in the LSCC study. This complexity reinforces the need to fully understand the mechanism and process of cell proliferation and differentiation. If scientists can control the pertinent transcription factors’ on-off switch like SOX2, it will not only reveal the pathway to potential cancer therapies, but also the ability to consistently create uniform differentiated cells. This is critical in testing new drugs for safety and efficacy, and ensuring that cells for transplantation are effective and safe.

References


1. National Institute of Health, Stem cell information. Visit: http://stemcells.nih. gov/info/basics/pages/basics1.aspx
2. Boumahdi S, SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma, Nature 511: pp246-250, 2014. Visit: www.nature.com/ nature/journal/v511/n7508/full/ nature13305.html
3. Justilien V et al, The PRKCI and SOX2 oncogenes are coamplified and cooperate to activate hedgehog signaling in lung squamous cell carcinoma, Cancer Cell 25(2): pp139-151, 2014. Visit: http://ac.elscdn. com/s1535610814000336/1-s2.0- S1535610814000336-main.pdf?_ tid=a2ce8be8-fc00-11e4-ba77-00000aab0f27&acdnat=1431804218_16492a67938967 f4c38000318e8fdbb8
4. Fields A and Justilien V, Interview on oncogenes driving squamous cell lung carcinoma, Cancernetwork. Visit: www. cancernetwork.com/lung-cancer/ oncogenes-driving-squamous-celllung- carcinoma
5. National Cancer Institute, Genetics of skin cancer – For health professionals (PDQ®). Visit: www.cancer.gov/types/ skin/hp/skin-genetics-pdq#link/ _41_toc
6. Santini R et al, SOX2 regulates selfrenewal and tumourigenicity of human melanoma-initiating cells, Oncogene 33(38): pp4,697-4,708, 2014. Visit: www.ncbi.nlm.nih.gov/pmc/articles/ pmc4180644
7. University of Toronto, Cell biologists discover on-off switch for key stem cell gene, ScienceDaily, 15 December 2014. Visit: www.sciencedaily.com/ releases/2014/12/141215084946.htm
8. Zhou HY et al, A Sox2 distal enhancer cluster regulates embryonic stem cell differentiation potential, Genes & Development 28(24): p2,699, 2014. Visit: http://genesdev.cshlp.org/ content/28/24/2699
9. On-off switch for critical stem cell gene discovered, Genetic Engineering & Biotechnology News, 15 December 2014. Visit: http://genengnews.com/ gen-news-highlights/on-offswitch- for-critical-stem-cellgene- discovered/81250699
10. University of Edinburgh, Stem cells reach standard for use in drug development, ScienceDaily, 11 June 2013. Visit: www.sciencedaily.com/ releases/2013/06/130611111712.htm
11. Cressey D, Stem cells take root in drug development, Nature, 2012. Visit: www. nature.com/news/stem-cells-take-rootin-drug-development-1.10713
12. Wyss Institute, Organs-on-chip, 2015. Visit: http://wyss.harvard.edu/ viewpage/461


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Regina Au is a Strategic Marketing Consultant at BioMarketing Insight, and has more than 20 years of experience in the biotech, pharma, medical device and diagnostic industries. She previously held sales and marketing roles at companies such as Merck, Genzyme Corp, NMT Medical and Radi Medical Systems. Regina has an MBA in Marketing from the University of Connecticut, a Microbiology degree from the University of Michigan, and a Master’s in International Management from Thunderbird School of Global Management.
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