Progress in Retinal and Eye Research 56 (2017) 19e31

Contents lists available at ScienceDirect

Progress in Retinal and Eye Research

journal homepage: www.elsevier.com/locate/prer

Retinal regeneration mechanisms linked to multiple cancer

molecules: A therapeutic conundrum
Amanda Barber a, 1, Kyle Farmer b, 1, Keith R. Martin a, c, d, 1, Patrice D. Smith a, b, *, 1
a
John van Geest Centre for Brain Repair, University of Cambridge, UK
b
Department of Neuroscience, Carleton University, Ottawa, Ontario, Canada
c
Medical Research Council e Wellcome Trust Cambridge Stem Cell Institute, Cambridge, UK
d
Cambridge NIHR Biomedical Research Centre, Cambridge, UK

a r t i c l e i n f o a b s t r a c t

Article history: Over the last decade, a large number of research articles have been published demonstrating regener-
Received 27 October 2015 ation and/or neuroprotection of retinal ganglion cells following manipulation of specic genetic and
Received in revised form molecular targets. Interestingly, of the targets that have been identied to promote repair following
6 March 2016
visual system damage, many are genes known to be mutated in different types of cancer. This review
Accepted 7 March 2016
Available online 30 August 2016
explores recent literature on the potential for modulating cancer genes as a therapeutic strategy for
visual system repair and looks at the potential clinical challenges associated with implementing this type
of therapy. We also discuss signalling mechanisms that have been implicated in cancer and consider how
Keywords:
Retinal ganglion cells
similar mechanisms may improve axonal regeneration in the optic nerve.
Axon regeneration 2016 Elsevier Ltd. All rights reserved.
Cancer genes
PTEN
Visual system repair mechanisms

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2. Signalling mechanisms involved in cancer and axon regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1. PI3K/PTEN/mTOR/Akt signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2. Cytokine signalling and inflammatory mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3. Anti-apoptotic factors and their role in cancer biology and implications for optic nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.4. Kruppel-like family (KLF) of transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.5. Rho/ROCK pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.6. Haematopoietically Expressed Homeobox (HHEX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.7. Epidermal growth factor receptor (EGFR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Abbreviations: AAV, Adeno-associated virus; CNS, Central Nervous System; CNTF, ciliary neurotrophic factor; DLK, dual leucine zipper kinase; DRG, dorsal root ganglion;
EGFR, Epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; GAP-43, Growth associated protein 43; GBM, glioblastoma multiforme; GM-CSF, gran-
ulocyte macrophage-colony stimulating factor; gp130, glycoprotein 130; HCC, Hepatocellular carcinoma cells; HHEX, Hematopoietically Expressed Homeobox; hnRNP K,
heterogeneous nuclear ribonucleoprotein K; IL-6, Interleukin-6; Jak/STAT, Janus kinase-signal transduce and activator of transcription; KLF, Kruppel-like Family; LIF, Leukemia
inhibitory factor; mTOR, mammalian target of rapamycin; NF-M, medium neurolament; PI3K, phosphatidylinositol-3-kinase; PTEN, phosphatase and tensin homolog; Rb,
retinoblastoma protein; RGC, retinal ganglion cell; SOCS-3, suppressor of cytokine signalling 3; Sfpq, Splicing factor proline- and glutamine-rich protein; STAT-3, signal
transducer and activator of transcription-3; VEGF, Vascular endothelial growth factor.
* Corresponding author. Department of Neuroscience, Carleton University, Ottawa, Ontario, Canada.
E-mail address: Patrice.Smith@carleton.ca (P.D. Smith).
1
Percentage of work contributed by each author in the production of the manuscript is as follows: Patrice D. Smith: 50%, Amanda Barber: 30%, Kyle Farmer: 10%, Keith
Martin: 10%.

http://dx.doi.org/10.1016/j.preteyeres.2016.08.003
1350-9462/ 2016 Elsevier Ltd. All rights reserved.
20 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31

3. Less explored potential targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.1. Heterogeneous nuclear ribonucleoprotein (hnRNP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3.2. Rb/E2F family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4. Approaches to reducing cancer risk when promoting regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5. Beyond axonal growth: challenges for repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
6. Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

1. Introduction the adult mammalian CNS. A major question is whether manipu-

lation of genes involved in cancer can be considered as a feasible
In recent years there has been an exponential increase in the mechanism to promote axon regeneration in a clinical setting,
number of publications reporting cell survival and/or axon regen- where the potential for oncogenesis is a concern. However, it is
eration in models of optic nerve injury, for example where intra- conceivable that the post-mitotic nature of neurons in the mature
ocular pressure is elevated to induce retinal ganglion cell (RGC) loss visual system may limit this risk of malignancy. In this review we
or following localised optic nerve crush. Numerous factors with will also consider how oncogenic risk might be mitigated through
diverse physiological functions, including mammalian target of careful selection and application of the pharmacological or mo-
rapamycin (mTOR), kruppel-like family (KLF) transcription factors, lecular tools used to manipulate cancer-associated pathways.
suppressor of cytokine signalling 3 (SOCS3) and cytokines (ciliary
neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)), have 2. Signalling mechanisms involved in cancer and axon
been suggested to play a key role in RGC survival and/or axon regeneration
regeneration. Proposed mechanisms include effects on the avail-
ability of neurotrophic factors, activation of intrinsic survival/ In this section we will focus on cancer genes and signalling
growth mechanisms, modulation of axonal transport and glia- mechanisms that have been proposed to underlie both malignancy
dependent support as well as inhibition of apoptotic mechanisms and regeneration in the mature nervous system.
in RGCs. Interestingly, many of the mechanisms that have been
suggested to promote either survival and/or axon regeneration in 2.1. PI3K/PTEN/mTOR/Akt signalling
the damaged visual system are directly or indirectly associated with
cancer, including malignancies that affect the eye and visual Many human cancers, including some tumours of the brain and
pathways. visual system, result from specic genetic alterations and there is
Cancer can be dened as the loss of normal cellular growth evidence that deletions in a particular region of chromosome 10 are
control where unregulated proliferation of cells result in malignant often involved (Kon et al., 1998; Li et al., 1997). As an example,
pathologies (De Potter et al., 2002). In contrast, failure of neuronal gliomas are the most common solid tumours in the CNS (brain,
regeneration can be considered as an intrinsic inability to re- spinal cord and visual system) and more than half of glioblastomas
activate the normal growth mechanisms that occur during devel- develop as a consequence of chromosome 10q loss (Bostrom et al.,
opment. In this sense, cancer and regenerative failure could be 1998). PTEN, a known tumour suppressor has been identied as a
viewed as different sides of the same coin. The central nervous candidate gene on chromosome 10q which is mutated in many
system (CNS) can be stimulated to regenerate by manipulation of a cancers and responsible for a large proportion of glioblastomas in
variety of cancer-associated molecular signalling pathways and the nervous system (Li et al., 1997).
excellent reviews have been published on this subject recently The PTEN protein is a 403 amino acid protein that is evolu-
(Benowitz et al., 2015; He and Jin, 2016; Kaplan et al., 2015; Smith tionarily conserved across species and through its many functional
et al., 2015). Of the identied regeneration-associated genes, many domains contributes to the regulation of a myriad of cellular
are tumour suppressors, including phosphatase and tensin homo- functions including cell survival, cellular growth, migration, pro-
log (PTEN (Park et al., 2008)) or oncogenes such as MYC (Buchser liferation, cellular homeostasis and metabolism (Fig. 1). PTEN
et al., 2010; Pomerantz and Blau, 2013). In this review, we will controls a number of complex cellular events at a transcriptional
focus on cancer genes and signalling mechanisms that have been level but also operates through post-translational modications of
proposed to underlie both malignancy and regeneration in the target proteins. Most well-known is its role as a regulator of the
mature nervous system. We will review several signalling path- PI3K pathway (Weng et al., 2001), which is mediated by the lipid
ways, including PTEN/mTOR pathway, phosphatidylinositol-3- phosphatase activity of PTEN that antagonises PI3K function in
kinase (PI3K), cytokine and inammatory factors including SOCS- many different physiological systems, including the brain. Inhibi-
3, trophic factors (such as CNTF), anti-apoptotic factors (including tion of PI3K, and downstream signalling via Akt, mTOR and S6 ki-
Bcl-2), KLF, transcription factors (including signal transducer and nase activity, results in arrested protein translation and cellular
activator of transcription-3 (STAT-3)) as well as additional proposed growth (Gao et al., 2000). Unsurprisingly, the phosphatase function
mechanisms that are known to be involved in cancer pathology but of PTEN is critical to its role as a tumour suppressor and loss of PTEN
that have also been implicated in axon regeneration. The optic function is known to play a role in initiating tumour pathogenesis
nerve crush model has been extensively utilized to evaluate the (Weng et al., 1999). Loss of the homeostatic and metabolic effects of
impact of modulating different signalling factors on axon regener- PTEN may also contribute to tumour formation (Cordero-Espinoza
ation in a physiological setting and has been important in dening and Hagen, 2013; Icard and Lincet, 2013). In addition, PTEN is
signalling mechanisms involved in promoting axon regeneration in intimately involved in regulating other tumour suppressor
 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31 21

molecules and as such loss of PTEN function could amplify the loss
of other tumour suppressors, potentiating the loss of control of
cellular growth. Overall, loss of PTEN function may result in
disruption of cell growth mechanisms, angiogenesis, cell prolifer-
ation and altered migration as well as dysregulation of other
tumour suppressor pathways.
PTEN is a potent suppressor of axon regeneration and several
groups have demonstrated enhanced axon regeneration of RGCs
(Park et al., 2008), mammalian peripheral sensory (sciatic) neuro-
nes (Christie et al., 2010) and corticospinal neurones (Liu et al.,
2010) following deletion or knockdown of PTEN, an effect mostly
mediated by enhancing mTOR signalling. PTEN deletion/enhanced
mTOR signalling has been shown to be important for axon regen-
eration in a number regeneration-competent species; examples
include Drosophila class IV dendritic arborisation (da) neurons
(Song et al., 2012b), C. elegans GABA motor neurons (Byrne et al.,
2014), zebrash dorsal root ganglion (DRG) neurons (Abe et al.,
2010) and blastemal cells following n amputation (Hirose et al.,
2014). The role of PTEN and mTOR signalling in axon regenera-
tion is functionally conserved across several highly divergent spe-
cies suggest that axon regeneration and the mechanisms that
regulate it are of ancient origin.
Previous work has shown that conditional gene deletion using
adeno-associated virus (AAV) mediated Cre deletion of oxed PTEN
in adult mouse RGCs is not only effective in promoting cell survival
but also initiates long distance axon growth in vivo (de Lima et al.,
2012; Park et al., 2008). Recently, Duan and colleagues demon-
strated that PTEN deletion specically promotes regeneration of a-
RGCs, a subtype of RGCs that preferentially survive post-axotomy
and show high mTOR signalling activity compared to other RGC

Fig. 1. PTEN is one of the most widely studied pathways in both retinal regeneration and cancer elds. PTEN is a 403 amino acid phosphatase that acts as a primary regulator of the
PI3k/Akt/mTOR signalling pathways. The amino terminus consists of a membrane binding domain that interacts with phosphoinositides. A core phosphatase domain is important in
facilitating the phosphatase activity of the protein. The C-terminal contains a C2 domain which is important in anchoring PTEN to specic protein targets as well as the plasma
membrane. The C-terminal domain is post-translationally regulated through phosphorylation events and contains binding sites for proteins containing PDZ-domains (A). The
expected folding pattern of the protein is represented in (B). PTEN blocks the conversion of PIP2 into PIP3 thereby preventing the activation of Akt. With feedback control by PTEN,
Akt regulates various intracellular events including cell survival, regeneration and cell death processes (C).
22 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31
subtypes (Duan et al., 2015). Further studies have shown successful
suppression of PTEN in adult wild type mouse corticospinal tract
axons using an RNAi (Zukor et al., 2013). This study demonstrated
that an shRNA-assisted approach to stimulating axon regeneration
of corticospinal tract axons following spinal cord injury recapitu-
lated the ndings of an earlier study by the same group (Liu et al.,
2010) whereby PTEN gene deletion resulted in axon regeneration
up to 2 mm beyond the lesion site. Geoffroy and colleagues recently
revealed an age-dependent decline in axon regeneration induced
by PTEN deletion in adult mammalian CNS neurons (corticospinal
and rubrospinal neurons) (Geoffroy et al., 2016). Interestingly, PTEN
deletion enhanced the intrinsic growth ability by increasing mTOR
activity in both young and aged CNS neurons, where neuronal soma
size and axonal growth proximal to the injury site was similar,
leading the authors to attribute the age-dependent decline in
regenerative ability to extrinsic inuences at and around the injury
site (Geoffroy et al., 2016). Whether the same relationship holds
true for RGCs remain to be explored.
Although the oncogenic potential of PTEN inhibition has not
been explored in regeneration studies, the involvement of PTEN in
several visual system and brain cancers suggests caution should be
adopted when manipulating PTEN for regenerative purposes.
Indeed, haploinsufciency of PTEN has been identied in many
tumours, including several which affect the visual system.
Approximately 70% of glioblastoma multiforme (GBM) cases show
loss of chromosome 10q (Bostrom et al., 1998), although it is
possible that other genes on 10q could also be involved in patho-
genesis of GBM. Clinical ndings suggest that alterations in the
PTEN gene (resulting in PTEN protein deciency) may contribute to
multiple aspects of glioma pathology and may play a critical role in
determining prognosis and therapeutic response in patients
suffering from this and other forms of cancer. Visual system gli-
omas, including retinoblastoma, are relatively common and
concerted research efforts have gone into identifying relevant
pathogenic mechanisms. Interestingly, many of the molecules that
have been implicated in axon regeneration processes in the visual
system have also been suggested to play a role in retinoblastoma.
Indeed, PTEN has been previously shown to be involved in the
control of retinoblastoma protein (Paramio et al., 1999), which is
critical in the development of retinoblastoma cancers; both spo-
radic and inherited forms of retinoblastoma have been linked to
PTEN (Zochodne, 2014). In addition to its proposed function in the
visual system, PTEN has also been linked to brain tumours
(Endersby and Baker, 2008; Haas-Kogan and Stokoe, 2008). This
supports the critical nature of PTEN function in regulating
cancerous growth in the CNS. The structural components of PTEN
are critical to its function in cellular growth (Fig. 1); in addition, the
downstream cascades that are involved in regulating PTEN function
can be PI3K-dependent or independent (Simpson and Parsons,
2001; Weng et al., 2001). To elaborate further on the PI3k inde-
pendent activity of PTEN it must be considered that the PTEN
protein is composed of 5 functional domains. Indeed, there are over
3000 reported mutations of the PTEN gene reported in the COSMIC
(catalogue of somatic mutations in cancer) online database (http://
cancer.sanger.ac.uk/cosmic) reecting the varied functions of PTEN
and its diverse role in cancer. PTEN acts as both a lipid phosphatase,
and is associated with mTOR pathway activation; it also acts as a
protein phosphatase when associated with focal adhesion kinase
(FAK) and plays a role in integrin signalling (Tamura et al., 1998,
1999b). Importantly, it is known to be involved in facilitating
tumour cells invasive and metastatic behaviours (Tamura et al.,
1998, 1999a). Indeed, the functions of PTEN have been expanded
signicantly in recent years to include phosphatase-independent
activities that have been elegantly reviewed by (Song et al.,
2012a). In addition, there may be potential unexplored functions
in the C-terminal domain of PTEN as several nonsense mutations
have been described in human tumours in the C-terminus of PTEN
outside of the traditionally studied phosphatase domains (Tamura
et al., 1999a, 1999b).
In summary, the pro-regenerative role of inhibiting PTEN func-
tion is now well established in the visual system through the use of
experimental animal models, particularly the optic nerve crush
injury model in mice. In addition, there is now ample evidence that
activating inammation (see below), combined with cAMP treat-
ment (Kurimoto et al., 2010) as well as genetic ablation of SOCS3
(Sun et al., 2011) may enhance the regeneration mediated by PTEN
inhibition. However, there is a conceivable oncogenic risk associ-
ated with manipulation of PTEN, a risk that could be associated
with the mTOR pathway itself or with non-mTOR-dependent,
PTEN-related activities. Perhaps oncogenic risk could be mini-
mised by developing an mTOR-specic method to facilitate axon
regeneration.
2.2. Cytokine signalling and inammatory mechanisms
Cytokines are inammatory messengers that play a critical role
in modulation of a variety of biological functions in the nervous
system. Advances in the eld of neuro-immunology have stim-
ulated efforts to understand the role of immune modulators in
nervous system function and several immunologically relevant
signalling cascades have been identied over the past two decades.
There are several examples of cytokines that promote axon
regeneration but also participate in signalling cascades relevant to
cancer.
Inammation, either as a result of lens injury or administration
of pro-inammatory agents such as zymosan, is known to promote
survival and regeneration of injured RGCs, an effect mediated by
both activated macrophages and retinal astrocytes (Leibinger et al.,
2009). Fischer and colleagues, demonstrated that upon inamma-
tory stimulation, CNTF, LIF, and interleukin-6 (IL-6) are upregulated
and released by retinal astrocytes and have pro-regenerative effects
on injured neurones via a signal transducer and activator of tran-
scription 3 (STAT3) dependent mechanism (Leibinger et al., 2013;
Muller et al., 2007). CNTF, IL-6 and LIF exert their effects by bind-
ing to receptors and forming a complex with glycoprotein 130
(gp130) (Heinrich et al., 2003; Leibinger et al., 2013). This complex
in turn activates the Jak/STAT (Janus kinase-signal transducer and
activator of transcription) pathway via phosphorylation of the
STAT3 transcription factor which initiates gene expression
(Heinrich et al., 2003). The gp130 complex can also trigger activa-
tion of the PI3K/mTOR pathway by phosphorylation of the cancer
associated protein tyrosine phosphatase, SHP-2 (Charest et al.,
2006; Hirano, 1998; Nicholson et al., 2000), supporting a role for
this pathway in cellular growth programs. SOCS-3 is a known Jak/
STAT signalling repressor and the ability of RGCs to respond to
CNTF, and thus regenerative ability, is greatly enhanced by SOCS3
deletion (Qin et al., 2013; Smith et al., 2009). Furthermore, RGC
axon regeneration can be observed in RGCs with constitutively
active STAT3 (Pernet et al., 2013). Recent work has demonstrated
extensive axon regeneration following transduction of RGCs with
an AAV construct expressing constitutively active Stat3CA fused
with viral active domain (VP16), which is known to hyperactivate
transcription factors by recruiting transcriptional co-factors to the
DNA binding domain (Hirai et al., 2010; Mehta et al., 2016). Inter-
estingly, in zebrash, a species that retains regenerative ability in
adulthood, Jak/STAT signalling driven in part by CNTF in the pres-
ence of SOCS3 (Smith et al., 2009) is important for robust optic
nerve regeneration (Elsaeidi et al., 2014; Smith et al., 2009). In
addition, LIF has been shown to be upregulated in zebrash RGCs 3
days after optic nerve injury and is immediately followed by STAT3
 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31
activation (Ogai et al., 2014).
Inammation is known to play a pivotal role in cancer initiation
and malignant progression, and Jak/STAT signalling has been
demonstrated to play a key role in the initiation of malignant
transformation and cancer progression of lymphopoiesis and
gastric adenomas (Jenkins et al., 2005; Yin et al., 2015). Indeed,
enhanced expression of the IL-6 cytokine superfamily, including
CNTF and LIF, has been demonstrated to be upregulated in human
brain tumours (Lilja et al., 2001). LIF has been shown to be over-
expressed in several types of cancers and has been associated with
the progression of several malignancies, including rhabdomyosar-
coma (cancer in striated muscle tissue), choriocarcinoma (cancer in
tissue that normally would become the placenta) and melanoma (Li
et al., 2014). As an inhibitory regulator of the Jak/STAT pathway,
SOCS3 can be described to have an anti-tumour effect and reduced
activity of SOCS3 has been associated with several cancers. For
example, hypermethylation has been attributed to silencing of
SOCS3 in human lung cancer (He et al., 2003). In addition, mouse
studies involving conditional SOCS3 knockout in either gastroin-
testinal epithelial cells (Inagaki-Ohara et al., 2014) or pancreatic
cells (Lesina et al., 2011) demonstrated an increase in tumour for-
mation. Although the tumour suppressor role of SOCS3 appears
clear, several studies indicate a varying role for SOCS3 depending
on the type of cancer. For example, SOCS3 knockdown in prostate
cancer resulted in enhanced apoptotic cell death (Puhr et al., 2009).
In addition, the number of lung and liver metastatic tumour nod-
ules was reduced in macrophage specic SOCS3 conditional
knockout mice compared to wild-type animals (Hiwatashi et al.,
2011). These studies indicate that the effect of SOCS3 dysregula-
tion can have varying effects depending on the tissue type. Indeed,
the long term effect of SOCS3 dysregulation within the visual sys-
tem is unknown, highlighting the need for cautious manipulation
of SOCS3 signalling pathways.
Recent work has revealed the potential benet of manipulating
granulocyte macrophage colony stimulating factor (GM-CSF) on
neurite growth in vitro (Hanea et al., 2016; Legacy et al., 2013). In
support of the current theme regarding the regenerative impact of
cancer-related molecules, GM-CSF is a hematopoietic cytokine that
is involved in regulating cancerous growth in several contexts
(Metcalf, 1985a,b). In particular, GM-CSF has the ability to regulate
the proliferation of myeloid leukemia cells (Metcalf, 1985a,b). Taken
together, the accumulating evidence points to a distinct role for
cytokine signalling in the regulating both axon regeneration and
cancer.
2.3. Anti-apoptotic factors and their role in cancer biology and
implications for optic nerve regeneration
Apoptosis is a form of programmed cell death which occurs
naturally in the nervous system and throughout the body. This
process is involved in regulating the number of cells in the devel-
oping nervous system and indeed of all developing tissue, but also
has a role in facilitating the removal of old and/or dying neurons.
Therapies that target the destruction of cancerous cells generally
trigger some form of cell death, including the induction of
apoptosis related mechanisms to eliminate cancerous cells. The
dysregulation of apoptotic mechanisms has been suggested as a
possible avenue to generate malignant cells (Mohammad et al.,
2015) where the uncontrolled proliferation caused by abnormal
apoptotic mechanisms results in cancer cell survival. In some in-
stances, this dysregulation of apoptosis could also contribute to
worse prognosis following therapeutic intervention. There are
many factors that orchestrate the apoptotic signalling cascade and
many potential molecular abnormalities can result from abnormal
function of these factors. Loss of several anti-apoptotic factors,
23
including Bcl-2, p53 and caspase 3, has been implicated in onco-
genesis (Sitorus et al., 2009). While these molecules have been
studied in the context of retinal cancers, it is of note that they have
also been modulated in models of retinal disease and have shown
promise as potential targets for therapeutic intervention. As an
example, Bcl-2 overexpressing mice show improved survival and
axon regeneration (Chen et al., 1997). Additionally, a supportive role
for Bcl-2 has been established in the retina (Huang et al., 2003).
Although it must be noted that the mood stabilizer, lithium, has
multiple mechanisms of action; in these experiments, lithium was
used to induce expression of Bcl-2, which in turn resulted in sig-
nicant improvement in RGC survival as well as a regenerative
effect on RGC axons (Huang et al., 2003). While normal retinal
tissue expresses low levels of Bcl-2, it was noted that specic
concentrations of lithium enhanced Bcl-2 expression and improved
RGC survival and axon regeneration. Intriguingly, and supporting
the idea that cancer-causing agents could be benecial to the
damaged visual system, previous work has demonstrated that
lithium treatment enhances the proliferation of potentially regen-
erative stem cells in the retina but is also associated with the
development of retinoblastoma (Silva et al., 2010). Studies exam-
ining the oncogenic risk of Bcl-2 manipulation in RGC axon
regeneration experiments are lacking.
Similarly, p53 has been repeatedly shown to be a major
contributor to cell death processes in the visual system with several
reports indicating the potential benet of reducing levels of p53 in
the damaged visual pathway (Park et al., 2008; Wilson et al., 2013).
The role of p53 in visual system repair remains controversial; our
work suggests that conditional deletion of p53 promotes RGC sur-
vival, but is not sufcient to enhance axon regeneration in vivo
(Park et al., 2008). P53 is a critical modulator of a variety of nervous
system functions and has an established role in growth inhibition
and DNA repair, where post-translational modication of the pro-
tein is critical to this latter function (reviewed in (Vousden and
Prives, 2009)). The pro-apoptotic functions of p53 have also been
actively investigated in both cancerous and non-cancerous tissue
types, with many studies supporting the role of p53 in cell death
mechanisms in the nervous system. The activation of p53 is highly
regulated and relies upon post-translational modication that fa-
cilitates its role as a transcription factor, with molecular targets of
diverse functions (Dai and Gu, 2010). While a possible role for
dysregulation of apoptotic molecules in cancer is well established,
the potential modulation of these factors as a possible means of
promoting repair of the damaged visual system deserves further
investigation. However the challenge remains in how to maximize
regeneration without increasing cancer risk.
Promoting cell death/apoptosis is not always detrimental to the
visual system. As an example, recent work suggests that modu-
lating mixed-lineage dual leucine zipper kinase (DLK) initiates a
cell death program which appears to be important in promoting
axon regeneration in the damaged visual system (Watkins et al.,
2013). In this regard, DLK was found to initiate a transcriptional
program which coupled the process of apoptosis to improvements
in axon regenerative ability in mice. While the mechanisms
involved are not absolutely clear, evidence does point to a distinct
role for DLK in regulating regeneration of RGC axons (Watkins et al.,
2013). A dramatic increase in expression of DLK was seen following
optic nerve crush injury (Watkins et al., 2013). The transcriptional
targets of DLK are not well known, but there are many genes that
have been identied as possible response elements for DLK. The
paradoxical induction of a presumed pro-apoptotic molecule, DLK,
with activation of regeneration associated genes emphasizes the
complexity of the problem that is to be targeted in promoting repair
of the damaged visual system. Nonetheless, again here the theme of
utilizing molecules implicated in oncogenesis remains. Indeed, DLK
24 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31
is considered a tumour suppressor and has been previously linked
with cancer development (Wu et al., 2010), although it has not yet
been identied in retinal cancers. The possible pro-apoptotic
function of DLK in malignant and non-malignant tissue has been
suggested and supports the possible utilization of this molecule as a
possible therapeutic target for cancer.
2.4. Kruppel-like family (KLF) of transcription factors
The Krppel-like family of transcription factors (KLFs) are a set
of zinc nger DNA binding proteins that regulate gene expression.
In mammals there are 17 genes in the KLF family expressed in
different tissues performing a variety of cellular functions from
proliferation to apoptosis, differentiation to migration and plurip-
otency. Many KLF members are expressed in neurons and recently,
it has been shown that specic KLF members can regulate intrinsic
axon growth ability of RGCs and other types of CNS neurons (Apara
and Goldberg, 2014; Moore et al., 2009), where different members
of the same KLF family have been shown to have opposing effects
on axon growth ability. The planarian atworm can regenerate any
missing body parts, and KLF has been shown to be required for
wound regeneration (Scimone et al., 2014) including photoreceptor
neuron regeneration (Lapan and Reddien, 2012). In mammalian
development the growth potential of axons diminishes with
maturation, a phenomenon that is associated with increased
expression of KLF4 and KLF9. Indeed, when these factors are
overexpressed in cell culture, neuronal axon growth potential is
inhibited (Moore et al., 2009). Furthermore, studies performed by
Moore et al. (2009) and Qin et al. (2013) demonstrated that axon
regeneration following optic nerve crush injury can be promoted by
genetic deletion or knock-down of KLF4 in adult mice, an effect that
was mediated by the Jak/STAT3 pathway (Moore et al., 2009; Qin
et al., 2013).
Conversely, KLF6 and KLF7 expression declines during the same
developmental time window where axon growth ability declines
illustrating the heterogeneity in function of the KLF family.
Furthermore, KLF6 and KLF7 expression have been shown to pro-
mote axon growth both in culture and in the regenerating optic
nerve of zebrash (Moore et al., 2009; Veldman et al., 2007). In
addition, Blackmore et al. (2012) demonstrated that in vivo acti-
vation of KLF7 enhanced axon regeneration in the spinal cord
(Blackmore et al., 2012).
Over the past few years evidence has emerged that KLF dysre-
gulation plays a role in human cancers, reviewed in detail by
Limame et al. (2014). Of the KLF members discussed above, KLF4,
whose inhibition promoted axon growth, has been described both
as a tumour suppressor and oncogene. However, there is a clear role
for KLF4 in cancer related epithelial-mesenchymal transition
(EMT), a process that involves a shift in cellular polarity enabling
cancer cells to invade the surrounding stroma. KLF4 has been
documented to be a potent inducer of epithelial differentiation in
EMT (Limame et al., 2014). KLF9, whose down regulation promotes
axon regeneration, surprisingly has been shown to positively
regulate p53 expression and has been identied as a tumour sup-
pressor in glioblastomas, colorectal tumours and hepatocellular
carcinoma (Sun et al., 2014). KLF6, whose expression results in
enhanced axon growth, has been described as a tumour suppressor
in prostate (Narla et al., 2001), hepatic (Zhenzhen et al., 2012) and
gastric cancer (Zhang et al., 2010) among others. Three splice var-
iants of the KLF6 gene have been documented which antagonise the
tumour suppressor function of wild type KLF6 (Narla et al., 2005).
However, the role of KLF6 in cancer is controversial. KLF6 has been
shown to be inactive in many solid tumours yet in vitro silencing of
KLF6 in hepatocellular carcinoma cells (HCC) induced apoptosis by
upregulating p53 and inhibiting pro-apoptotic Bcl-xL expression,
highlighting that KLF6 is essential for HCC cells to evade cell death
(Sirach et al., 2007). Studies to examine how KLF6 exerts its pro-
regenerative effect would be interesting, especially to identify
whether axon growth is associated with enhanced p53 expression.
Expression of KLF7, which has been demonstrated to support axon
growth, has been associated with a poor prognosis of paediatric
acute lymphoblastic leukemia and is used as a biomarker for
therapy resistance and relapse (Schuettpelz et al., 2012). Although
the mechanism behind this phenomenon is not clear, it has been
suggested that enforced expression of KLF7 resulted in upregula-
tion of pro-survival members of the Bcl2 family, whose expression
has been associated with chemotherapy resistance (Schuettpelz
et al., 2012). It would be interesting to examine whether the pro-
regenerative effects of KLF7 were accompanied by enhanced sur-
vival of neurones and mediated by Bcl2.
The role of KLFs in cancer are varied and although the long term
effect of KLF manipulation within a regenerative context has not
been explored, the lack of associated KLFs to visual system related
malignancies may suggest the oncogenic risk is low. Instead, the
cancer literature may offer us clues as to how KLF manipulation
may promote axon regeneration.
2.5. Rho/ROCK pathway
ROCK (Rho-associated protein kinase) is the downstream sig-
nalling protein of the small GTPase Rho, both of which form the
Rho/ROCK (Rho/Rho-associated coiled-coil containing protein ki-
nase) pathway, an important signal transduction system within the
CNS. Most commonly known for regulating the cytoskeleton and
playing an essential role in cell migration, growth and development
(Chen and Yao, 2013), the Rho/ROCK pathway is also important in
regulating visual system function by synthesizing extracellular
matrix components, critical for the normal function of the retina
(Watanabe, 2010). Furthermore, Rho/ROCK mechanisms have been
implicated in maintenance of intraocular pressure, with the
development of ophthalmic solution to treat glaucoma based upon
Rho/ROCK pathway inhibition (Kaneko et al., 2016; Wang et al.,
2013). The role of the Rho/ROCK pathway in regeneration-
competent species has not been examined in great detail. Previ-
ous work has shown that the Rho/ROCK pathway is required for
interkinetic nuclear migration of proliferating Muller glia that
drives regeneration of photoreceptors in zebrash, a process that
does not translate to mammals (Lahne et al., 2015). The most
informative study is offered by Beane and colleagues, who
demonstrated a role for ROCK in planarian atworm regeneration
(Beane et al., 2012), where inhibition of ROCK by RNAi led to
excessive growth of optic neurons well after control animals had
nished their regeneration process. This suggests a role for the Rho/
ROCK pathway as a tissue growth limiting mechanism to reduce
production and replacement of cells and prevent uncontrolled
growth. Although the long term effects of Rho/ROCK inhibition in
mammalian regeneration has not been examined, the Beane study
may indicate that long term inhibition is undesirable (Beane et al.,
2012).
The potential role of the Rho/ROCK pathway in CNS regenera-
tion is reviewed extensively previously (Liu et al., 2015; Wang
et al., 2013). The adult CNS offers a hostile environment to
regenerating axons, through the expression of myelin-derived
inhibitory molecules (Nogo, OMgp, MAG), chondroitin sulphate
proteoglycans that form components of the glia scar (Fawcett,
2006; Smith et al., 2015) and inhibitory guidance cues such as
semaphorins and ephrins (Benson et al., 2005). These extracellular
factors exerts their regeneration inhibitory effects through acti-
vation of their specic surface receptors on axons (eg Nogo re-
ceptor, paired immunoglobulin-like receptor, p75 neurotrophin
 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31 25

receptor, Lingo-1) but their signalling converges by activating the
Rho/ROCK pathway which modulates the actin cytoskeleton and
results in growth cone collapse and inhibition of neurite out-
growths. Indeed, RhoA, ROCK1 and ROCK2 have been shown to be
expressed at the optic nerve head where they may act to inhibit
axon regeneration (Goldhagen et al., 2012). Inhibition of the Rho/
ROCK signalling pathway has thus received much attention in
efforts to promote regeneration of injured axons. Previous
research has demonstrated that through targeting of the small
GTPase Rho, regeneration of RGCs can be enhanced in vitro
(Monnier et al., 2003) and in adult rodents following optic nerve
lesion by overcoming myelin inhibition (Bertrand et al., 2005;
Fischer et al., 2004). The most commonly known inhibitor of the
Rho/ROCK pathway is Y-27632 which acts by blocking the ATP-
binding site on the catalytic domain of ROCK. (Zohrabian et al.,
2009). Lingor and colleagues demonstrated that Y-27632, along
with other ROCK inhibitors Fasudil (HA-1077) and Dimethylfasudil
(H-1152), promoted neurite outgrowth of RGCs cultured on
inhibitory chondroitin sulphate proteoglycan substrate (Lingor
et al., 2007). However, it is worth mentioning that these widely
used ROCK inhibitors can have multiple targets and can inhibit a
number of other kinases at high doses (Anastassiadis et al., 2011).
Y-27632 also stimulated RGC axon regeneration in the rat optic
nerve crush in vivo model in a dose dependent manner (Lingor
et al., 2007) and in adult cats (Sagawa et al., 2007). Interestingly
combined ROCK inhibition with increased CNTF results in additive
effects on neurite outgrowth extension in vitro (Ahmed et al.,
2009) and on RGC axon regeneration following optic nerve crush
in adults rats (Lingor et al., 2008). Similarly co-administration of Y-
27632 with cAMP (Ahmed et al., 2009) and erythropoietin (Tan
et al., 2012) also boosted the regenerative effects. Y-27632 alone
has also been shown to increase myelination in an in vitro injury
spinal cord model (Boomkamp et al., 2012), a phenomenon that
has been shown to be important in optic nerve regeneration (Bei
et al., 2016), however, whether Y-27632 can promote re-
myelination of the optic nerve, or in in vivo models, remains to
be addressed.
The Rho/ROCK signalling pathway is very well established in
oncogenesis (Pruitt and Der, 2001) and the ways in which it con-
tributes to the oncogenic process are numerous and only briey
reviewed here (and illustrated in Fig. 2). For a more detailed ac-
count, comprehensive reviews can be found from (Chin et al., 2015;
Rath and Olson, 2012). As mentioned above, the Rho/ROCK
pathway is central to regulation of the actinomycin cytoskeleton
and thus to cell motility, a function that is essential for cancer cell
migration and invasion. Indeed aberrant activation of this pathway
is well established in the literature as a potential precursor to
migration and invasion of tumour cells in vitro and in vivo (Itoh
et al., 1999; Li et al., 2006). In addition, the Rho/ROCK pathway is
critical for tumour-associated angiogenesis, which is a key char-
acteristic of tumour progression and is stimulated by the intra-
tumour hypoxic environment and largely controlled by vascular
endothelial growth factor (VEGF)-mediated Rho/ROCK activation
(Chin et al., 2015). Rho/ROCK signalling also plays a role in loos-
ening the endothelial intercellular junctions, a process required for
the regulation and development of new endothelial capillaries (Liu
and Senger, 2004).
Many studies have demonstrated the therapeutic value of
inhibiting the Rho/ROCK pathway in cancer models, including
reduced cellular proliferation, invasion and angiogenesis in vitro

Fig. 2. The Rho/ROCK pathway is a critical regulatory cascade that is primarily involved in regulating cytoskeletal structure and maintenance, while playing a critical role during
early development. Importantly, the Rho/ROCK pathway has also been found to be a key regulator of PTEN signalling mechanisms. As such, this pathway is often studied in the
context of both regeneration and oncogenesis. Y-27632 has several kinase targets; it is an effective inhibitor of ROCK signalling.
26 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31
and reduced tumour growth and metastasis in vivo which are
reviewed extensively by Chin et al. (2015) and too numerous to
discuss in detail in this review (Chin et al., 2015).
In summary, considering the varied role of the Rho/ROCK
pathway in cell migration and adhesion it is of no surprise that it
has an important role in both axon regeneration and oncogenesis.
What we can learn from the cancer literature is that there are
numerous mechanisms by which the Rho/ROCK pathway can
contribute to the oncogenic process, unsurprising considering the
plethora of affectors and effectors of this pathway. The specic
details of how the Rho/ROCK operates in oncogenesis are more
thoroughly understood in comparison to the axon regeneration
literature. It would be interesting to have a clearer picture of which
upstream affectors and downstream effectors of the Rho/ROCK
pathway account for the regeneration effects. Rho/ROCK dysregu-
lation promotes later stage cancer processes and whether
manipulation of this pathway is enough to cause cancer is unclear.
The oncogenic risk of Rho/ROCK pathway inhibition as a method to
stimulate axon regeneration may therefore be minimal, but when
used in combinatorial therapies such as PTEN gene deletion etc, the
oncogenic risk could conceivably be considerably elevated. Indeed
little is known about the role of Rho/ROCK in regeneration
competent species, although there is evidence that points to the
Rho/ROCK pathway acting as a regenerating tissue growth brake
that may minimize oncogenic risk.
2.6. Haematopoietically Expressed Homeobox (HHEX)
Recently, Blackmore and colleagues tested a large number of
transcription factors that have previously been implicated in
regulating tumour suppressive functions in neuronal cells and
evaluated the impact of modulating these factors on neurite growth
(Simpson et al., 2015). A functional screen identied several genes
that were considered tumour suppressor or oncogenic in regulation
of neurite growth. Of the molecules identied, the authors carried
out follow-up experiments to determine the impact of modulating
the CNS expressing tumour suppressor, HHEX (Su et al., 2012), on
axon growth ability. This work demonstrated that overexpression
of HHEX reduced axon growth ability, supporting a role for HHEX in
limiting axon growth in the CNS (Simpson et al., 2015). These data
again support the theme and hypothesis that repurposing and
Fig. 3. Throughout this review we have discussed various genes, receptors, ligands, and messenger systems, and how they contribute to either regenerative and/or oncogenic
processes as individual contributing factors. Importantly, the majority of the factors discussed act on complimentary systems and can be combined into a comprehensive pathway
with signicant crosstalk among signalling factors. When taken together one can easily see how these factors interact with each other and all to cell survival, regeneration as well as
cellular proliferation.
unlocking the role of oncogenes and tumour suppressors could be
effective in facilitating axon growth in the adult CNS, including the
visual system.
2.7. Epidermal growth factor receptor (EGFR)
The EGFR gene mutations are frequently seen in lung cancers,
particularly in adenocarcinomas (Midha et al., 2015). EGFR is well
established to be involved in the pathogenesis of cancer, particu-
larly in the lung, although there is currently little evidence sup-
porting a role for EGFR in cancers of the visual system. However,
EGFR has been suggested to play a critical role in neuronal function
throughout the CNS. EGFR is expressed in the retina, where it
regulates neuronal as well as glial precursor proliferation and dif-
ferentiation (Chen et al., 2007). The function of EGFR in mature
neurons is not well dened, but there is evidence suggesting a
possible role in regulation of RGC survival and axon regeneration
(Berry et al., 2011). EGFR transactivation has been suggested to play
a key role in inhibition of CNS axon growth. In this regard, small
molecule inhibitors of EGFR promote neurite growth on inhibitory
substrates in vitro and axon regeneration after optic nerve injury
in vivo (Koprivica et al., 2005). However, the role of EGFR in the
visual system remains unclear (Berry et al., 2011; Koprivica et al.,
2005). A proposed role for EGFR in regulating RGC viability in a
model of retinopathy has been suggested by Hewing et al. (2013),
where the authors showed that pharmacological EGFR inhibition
protects RGCs from oxygen-induced retinopathy in mice (Hewing
et al., 2013). However, it must be considered that inhibitors of
EGFR can have dose dependent off-targets effects, including ERBB4
and other kinases, as such these effects may be a combined result of
multiple receptor systems and not solely EGFR. Although the evi-
dence remains controversial, the theme holds true for EGFR which
represents another oncogenic molecule that could be targeted to
promote repair of the damaged visual system.
3. Less explored potential targets
Manipulating cancer-associated genes to promote repair and
recovery in the damaged CNS may appear counter-intuitive, but it is
worth noting that nature uses these growth mechanisms for pre-
cisely this purpose in other organisms while avoiding oncogenesis.
 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31
Indeed some non-mammalian species, namely zebrash, xenopus
and other anamniotes, retain the capacity to regenerate CNS axons
well into the adult years (Turner and Delaney, 1979). Several factors,
discussed in this section, have been identied to contribute to the
regenerative process in these models that have yet to be explored in
mammalian regeneration. We will also discuss other unexplored
targets generated by mammalian research.
3.1. Heterogeneous nuclear ribonucleoprotein (hnRNP)
Both growth associated protein 43 (GAP-43) and medium neu-
rolament (NF-M) are needed to establish the growth cone and
facilitate axon elongation and thus are important for axon regen-
eration (Jacobson et al., 1986). Expression of both is tightly
controlled by transcriptional and post-transcriptional methods.
RNA-binding proteins regulate the translation of specic RNAs.
RNA-binding protein hnRNP K directly binds both GAP-43 and NF-
M RNAs exporting them from the nucleus for translation and has
been shown to be essential in Xenopus optic axon regeneration (Liu
et al., 2012). The role of hnRNP K in mammalian axon regeneration
has yet to be explored but caution must be adopted as it this RNA
binding protein has been described as both a tumour suppressor
and oncogene (Gallardo et al., 2016).
3.2. Rb/E2F family
The retinoblastoma protein (Rb1) is involved in regulating cell
cycle progression and suppresses E2F transcriptional activity to
promote its cellular functions. Rb phosphorylation regulates and
inhibits its binding to E2F. Rb1 is well characterized as a gene that is
frequently deleted in retinoblastoma, which is seen in young chil-
dren (Friend et al., 1986). The mechanisms utilized by Rb are not
clearly understood, and currently there is no clear evidence sup-
porting a role for RB in optic nerve regeneration, in contrast to
PTEN, where PTEN decient neurons are effective at mounting a
regenerative response in the visual system (Park et al., 2008; Smith
et al., 2009; Sun et al., 2011). Interestingly, co-deletion of both PTEN
and Rb results in the development of retinoblastoma (Xie et al.,
2015). Furthermore, the lysine acetyltransferase p300, which is
also linked to retinoblastoma has also been implicated in enhancing
regenerative axonal growth (Gaub et al., 2011). Overexpression of
p300, which is normally involved in development of RGCs, plays a
key role in promoting axon regeneration following optic nerve
damage (Gaub et al., 2011). Together, these data support a possible
role for retinoblastoma genes in regulating axon regeneration
mechanisms in the adult visual system, but further investigation is
necessary to clearly dene this potential role.
4. Approaches to reducing cancer risk when promoting
regeneration
Neuronal regenerative therapies that target cancer eassociated
molecular pathways raise a serious concern regarding oncogenesis,
although conceivably this risk may be limited by the post-mitotic
nature of the mature visual system. Genes may function differ-
ently in a post-mitotic cellular context, thereby allowing the
modulation of tumour suppressors without enacting oncogenic
mechanisms. Thus it is plausible that modulation of tumour sup-
pressors, if targeted in a spatially and temporally directed manner,
could be benecial to repair and recovery of the CNS, including the
visual system. There are multiple options available that could
facilitate the modulation of cancer molecules to promote axon
regeneration in a clinically safer manner. For example viral-
mediated options have been consistently suggested, where the vi-
rus is deemed replication decient and expression can be limited
27
both by serotype tropism and by the use of a cell specic promoter.
However non-viral approaches such as siRNA and small molecule
or peptide compounds could also be of particular importance
where the temporal effect is limited by their degradation rate.
Small molecule inhibitors of tumour suppressor genes or activators
of oncogenes could be feasible therapeutically as they can be
delivered locally to the eye, where the retinal blood barrier may
restrict spatial delivery avoiding unwanted systemic effects.
As mentioned previously, in other regeneration competent
model systems cancer-associated genes have been identied to
promote repair and recovery in the damaged CNS while avoiding
oncogenic risk. Studies examining how cancer-associated genes
and pathways are used in regeneration competent model systems
may inform us of how to manipulate these genes/pathways safely
in the mammalian system. For example PTEN/mTOR activity has
been associated with many cancers and has been demonstrated to
facilitate neuronal axon regeneration in rodents (Park et al., 2008;
Smith et al., 2009; Sun et al., 2011). mTOR activity has also been
shown to be important for regenerating tissues in the zebrash,
specically in DRG neurons (Abe et al., 2010), blastemal cells
following n amputation (Hirose et al., 2014), and RGCs post optic
nerve injury (Diekmann et al., 2015). Interestingly, all tissues
exhibit a transient burst of mTOR activity followed by rapid down
regulation. Transient mTOR activity can be observed in zebrash
RGCs up to 4 days post optic nerve injury correlating with RGC
axonal growth, an effect that was compromised by mTOR inhibition
by rapamycin (Diekmann et al., 2015). It has been suggested that
activation of the mTOR pathway post axotomy in zebrash is tightly
regulated by expression of LIF (Diekmann et al., 2015; Ogai et al.,
2014) and by its autoregulatory negative feedback mechanism
where activated mTOR itself strongly represses the PI3K/AKT
pathway (Harrington et al., 2005). LIF also shows a similar time
course of expression as mTOR activity in the zebrash axotomy
model, with an early transient peak of expression followed by a
quick down regulation (Ogai et al., 2014). Such tight spatial regu-
lation of both mTOR activity and LIF expression may help to subvert
uncontrolled cellular growth and thus oncogenic risk.
In addition, Jak/STAT signalling regulated in part by CNTF drives
optic nerve regeneration in the zebrash even in the presence of
SOC3 (Elsaeidi et al., 2014). Interestingly, SOCS3a and splicing factor
proline- and glutamine-rich (Sfpq) proteins known to inhibit the
regenerative response, were upregulated following optic nerve
lesion and regulated by Jak/STAT signalling (Elsaeidi et al., 2014).
Knockdown of these proteins enhanced optic nerve regeneration,
as can also be observed in the mammalian system. Most intrigu-
ingly, zebrash were able to overcome the inhibitory effects of
SOCS3a and Sfpq but inducing IL-6 cytokines expression, including
CNTF, IL-11 and Clcf1/Crlf1a, in RGCs to stimulate Jak/Stat signalling
leading to robust axon regeneration. This suggests that expression
of SOCS3 may act as an inhibitory mechanism to prevent uncon-
trolled and aberrant axonal growth. These studies indicate that it is
possible to overcome Jak/STAT signalling suppression by SOCS3
without genetic deletion and that perhaps a combinatorial
approach using several cytokines may be of use to drive Jak/STAT
mediated axon regeneration. An alternative approach is to adopt
the use of designer growth factors to induce a stronger and more
specic regeneration effect than natural growth factors. A good
example of such an approach was recently reported by Leibinger
et al. (2016), whereby a designer cytokine hyper-IL-6 (hIL-6) was
delivered virally to directly activate the gp130 receptor (Leibinger
et al., 2016). The authors demonstrated stronger activation of JAK/
STAT3 and PI3K/Akt/mTOR signalling pathways, which was
accompanied by increased neurite growth of cultured RGCs and
DRGs and enhanced axon regeneration in the optic nerve crush
mouse model compared to CNTF and IL-6. This study highlights the
28 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31

Table 1
The role of various factors in retinal regeneration and/or carcinogenesis. The table summarises selected sources referenced for each factor proposed to be involved in regulating
both axon regeneration and cancer mechanisms.

Factor Retinal regeneration sources Cancer sources

Bcl-2 Chen et al., 1997; Huang et al., 2003 Schuettpelz et al., 2012; Sitorus et al., 2009
DLK Watkins et al., 2013; Wu et al., 2010
EGFR Berry et al., 2011; Chen et al., 2007; Hewing et al., 2013; Koprivica et al., Midha et al., 2015
2005
GM-CSF Hanea et al., 2016; Legacy et al., 2013 Metcalf, 1985a,b
HHEX Simpson et al., 2015 Su et al., 2012
hnRNP K Liu et al., 2012 Gallardo et al., 2016
JAK/STAT Elsaeidi et al., 2014; Moore et al., 2009; Smith et al., 2009; Qin et al., 2013 Jenkins et al., 2005; Yin et al., 2015
KLF Apara and Goldberg, 2014; Lapan and Reddien, 2012; Moore et al., 2009; Limame et al., 2014
Scimone et al., 2014
KLF4 Moore et al., 2009; Qin et al., 2013 Limame et al., 2014
KLF6 Moore et al., 2009; Veldman et al., 2007 Narla et al., 2001, 2005; Zhang et al., 2010; Zhenzhen et al., 2012
KLF7 Blackmore et al., 2012; Moore et al., 2009; Veldman et al., 2007 Schuettpelz et al., 2012;
KLF9 Moore et al., 2009 Sun et al., 2014
LIF Diekmann et al., 2015; Leibinger et al., 2009; Muller et al., 2007; Ogai et al., Li et al., 2014
2014
mTOR Abe et al., 2010; Charest et al., 2006; Diekmann et al., 2015; Hirano, 1998; Park et al., 2008
Hirose et al., 2014; Nicholson et al., 2000; Ogai et al., 2014; Park et al., 2008
MYC Belin et al., 2015 Weiss, 1982
p53 Dai and Gu, 2010; Park et al., 2008; Vousden and Prives, 2009; Wilson et al., Sirach et al., 2007; Sitorus et al., 2009; Sun et al., 2014
2013
PTEN Abe et al., 2010; Byrne et al., 2014; Christie et al., 2010; de Lima et al., 2012; Bostrom et al., 1998; Cordero-Espinoza and Hagen, 2013; Endersby and
Duan et al., 2015; Geoffroy et al., 2016; Hirose et al., 2014; Liu et al., 2010; Baker, 2008; Haas-Kogan and Stokoe, 2008; Icard and Lincet 2013; Li et al.,
Park et al., 2008; Smith et al., 2009; Song et al., 2012a,b; Sun et al., 2011; 1997; Paramio et al., 1999; Weng et al., 1999; Xie et al., 2015; Zochodne,
Zukor et al., 2013 2014
Rb1 Park et al., 2008; Smith et al., 2009; Sun et al., 2011 Friend et al., 1986; Xie et al., 2015
Rho Bertrand et al., 2005; Chen and Yao, 2013; Goldhagen et al., 2012; Kaneko Chin et al., 2015; Itoh et al., 1999; Li et al., 2006; Liu and Senger, 2004; Pruitt
et al., 2016; Lahne et al., 2015; Monnier et al., 2003; Wang et al., 2013; and Der, 2001; Rath and Olson, 2012
Watanabe, 2010
ROCK Ahmed et al., 2009; Beane et al., 2012; Bertrand et al., 2005; Boomkamp Chin et al., 2015; Itoh et al., 1999; Li et al., 2006; Liu and Senger, 2004; Pruitt
et al., 2012; Bei et al., 2016; Chen and Yao, 2013; Kaneko et al., 2016; Lahne and Der, 2001; Rath and Olson, 2012
et al., 2015; Lingor et al., 2008; Liu et al., 2015; Monnier et al., 2003; Tan
et al., 2012; Wang et al., 2013; Watanabe, 2010; Zohrabian et al., 2009
SOCS-3 Elsaeidi et al., 2014; Smith et al., 2009; Sun et al., 2011; Qin et al., 2013 He et al., 2003; Hiwatashi et al., 2011; Inagaki-Ohara et al., 2014; Lesina
et al., 2011; Puhr et al., 2009

importance of understanding the expression proles of growth
factor receptors. Specically, both CNTF and IL-6 must rst bind to
their non-signalling a-receptors, which in turn acts to recruit
further signalling receptor subunits: gp130/LIFR in the case of CNTF
and a gp130 homodimer in the case of IL-6 (Heinrich et al., 2003).
Low expression of the a-receptor subunits rather than gp130 itself
results in a limited response to CNTF or IL-6 (Heinrich et al., 2003),
indeed in the case of optic nerve crush injury the CNTF a-receptor
subunits is actually downregulated (Miotke et al., 2007). In this
instance, direct activation of the gp130 receptor subunit would be
favourable and the designer hIL-6, that consists of the covalently
linked bioactive parts of IL-6 and soluble IL-6a, does exactly this
(Peters et al., 1998). The development of designer growth factors
may offer a method to specically and more effectively activate
specic elements of the relevant signalling pathways involved in
axon regeneration.
As an alternative strategy, it is conceivable that toxin or drug
sensitivity could be engineered into cells as part of a regenerative
therapy to enable cells to be selectively eliminated should they
escape cell cycle control. Conditional gene expression systems,
where genes can be turned on or off using drugs such as tamoxifen
or tetracycline, have been widely used in neuroscience research and
could potentially form part of a risk reduction strategy for neural
regeneration treatments (Schonig et al., 2012).
5. Beyond axonal growth: challenges for repair
In order to promote repair and recovery following CNS damage,
neurons must survive and then extend regenerating axons, which
must then traverse the hostile inhibitory CNS environment and nd
appropriate synaptic targets to allow functional circuits to be re-
established. However manipulating factors which promote axonal
growth may not be sufcient to restore functions such as useful
vision. In addition, the therapeutic time window for axonal
regeneration will need to be established and the extent of target
CNS remodelling post injury should be considered when evaluating
re-connectivity of visual input. We have previously shown that
manipulation of some cancer-related genes, such as PTEN and
SOCS3, promotes both neuron survival and axon regeneration
in vivo (Park et al., 2008; Smith et al., 2009). Others have also shown
enhanced axonal regeneration by manipulating other cancer-
related mechanisms, either with (Belin et al., 2015) or without
(Moore et al., 2009) enhanced cell survival. Our previous work also
found evidence of dissociation between cell survival mechanisms
and axon regeneration mechanisms in the visual system in certain
contexts. We have shown that p53, a well-known tumour sup-
pressor, when conditionally knocked out in RGCs, promotes neuron
survival but has no effect on axon regeneration (Park et al., 2008).
Together, these results support the claim that combinatorial ap-
proaches to protection and regeneration may be necessary to
facilitate recovery following CNS injury, especially given the po-
tential molecular crosstalk among the different factors (Fig. 3).
Indeed, some of the cancer regulatory molecules that have been
discussed throughout this review, show dramatic impact on not
only axon regeneration, but in most cases cell survival as well. The
possibility to harness the modulation of oncogenes and tumour
suppressor molecules toward promoting both cell survival and
axon regeneration in the post-mitotic neurons of the retina is of
particular interest and value as we strive to identify molecular
avenues to promote repair of the damaged visual system.
 A. Barber et al. / Progress in Retinal and Eye Research 56 (2017) 19e31
6. Conclusion and future directions
It is clear that where most extensive axon regeneration has been
observed in the adult CNS to date it has resulted from manipulation
or indirect regulation of cancer associated pathways (see Table 1 for
summary of selected factors discussed here). Many of these studies
have exclusively focused on the regenerative potential of manipu-
lated cancer associated genes but it is clear that the long term
oncogenic risk will need to be assessed if such therapies can be
considered as a realistic option for patients. Several considerations
must be taken into account to effectively implement this type of
therapeutic approach. Certainly cancer gene manipulation will
require consideration of both spatial and temporal constraints to
reduce the possibility of oncogenesis in the adult CNS.
Even in the event that there is no therapeutically appropriate
avenue to implement cancer gene therapy as a mechanism to
promote regeneration, there is tremendous value in the under-
standing gained from studying the role of these molecules in the
CNS. The knowledge gained could provide important insights into
the cellular and molecular mechanisms required to promote axon
regeneration, thereby driving additional research into which mo-
lecular targets are critical to promoting regeneration in the adult
CNS.
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