Association of Squamous Cell Carcinoma and Hyaluronan: A Scope of the Literature

Nonmelanotic skin cancer (NMSC) refers to 2 main groups of tumors: cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC).¹ The most commonly diagnosed form of cancer globally, NMSC accounts for nearly one-third of all malignancies.² NMSC is ranked 24th in terms of highest mortality of all types of cancer.³ Over the past 30 years, cSCC incidence has increased by 3% to 10% annually.⁴ Although cSCC has a relatively low mortality rate, due to the high number of cases, millions of people are lost to the disease annually.⁵

The clinical presentation of a cSCC lesion can vary from premalignant lesions (actinic keratosis [AK]) to squamous cell carcinoma (SCC) in situ (Bowen’s disease [BD]) or invasive cSCC with the possibility of distant metastasis.^6,7 Several etiological factors are associated with developing SCC, such as ultraviolet (UV) radiation (which is widely reported as the most prevalent risk factor for NMSC), older age, ionizing radiation, chemical carcinogens, viruses, chronic inflammation, and immunosuppression.^8-10 The above environmental factors, along with an underlying genetic susceptibility associated with male sex, light-colored eyes, and red or blonde hair, considerably increase an individual’s lifetime risk.¹¹

The mainstay of treatment for SCC is surgical excision or radiotherapy for nonsurgical candidates.¹² Other treatments are available for cSCC, including retinoids, chemotherapy agents, photodynamic therapy, and topical agents, all of which have been associated with varying degrees of success when used in isolation versus when paired together.¹² Following diagnosis and treatment, the majority of patients require constant surveillance as a suspicious lesion rarely presents as a single event without future occurrences.¹³ Once treated, the recurrence rate will vary based on the original site, depth, diameter, and severity of the cSCC.⁵ The mortality rate of cSCC is often underreported; however, if cancer has progressed to distant metastasis, the 5-year mortality rate is 89%.⁷ Further, immunosuppressed patients following organ transplants or exposure to certain viruses have a 60 to 250 times greater risk of developing cSCC, particularly a much more aggressive subtype of cSCC.^7,14

The skin, otherwise known as the integument, is the largest organ of the human body, regulating many processes while providing protection, sensation, and circulation.¹⁵ The skin comprises 3 distinct layers (epidermis, dermis, and hypodermis) that can be subdivided further.¹⁶ The epidermis as a whole is divided into 5 layers: the stratum basale (deepest), stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum (most superficial).¹⁶

These layers are susceptible to damage, which may result in the development of nonmalignant, premalignant, and malignant lesions. These lesions can only present in a handful of ways on the human body, and as such, the differences between a benign and malignant lesion can be very subtle and often indistinguishable to the naked eye.¹⁷ Some lesions that are considered benign have the potential to become malignant if left untreated.¹⁸ The most common forms of these lesions, known as keratinocyte-derived tumors, are AK, BD, and keratoacanthoma (KA).¹⁹

The development and progression of cSCC is highly dependent on a person’s genetics.²⁰ There have been many factors linked with the development of cSCC; however, UV radiation is the most notable culprit.²⁰ UV radiation, particularly the cumulative effect of UVB radiation and also UVA radiation in a susceptible individual, is now considered the main etiological factor in developing the majority of benign and malignant skin lesions.²¹ Other significant factors, such as chemical or mechanical trauma, chronic inflammation, immunosuppression, viruses, and certain hereditary and medical conditions, have all been linked to an increased risk of developing cSCC.²¹

The diagnosis of cSCC is based primarily on clinical features and suspicion; however, histological analysis of a lesion remains the most significant factor in staging and guiding treatment.¹² Clinical features that would suggest a cSCC include a hyperkeratotic lesion that can be painful or ulcerated; dermoscopy can reveal vascular patterns, brown globules, and a gray-brown homogeneous pigmentation.^5,12 cSCC can present in a multitude of ways, thus emphasising the importance of obtaining a biopsy when there is increased suspicion.¹² Diameter, depth, perineural involvement, previous history of skin cancer, location, previous trauma to the area, and lymph node involvement all contribute heavily to the treatment plan and prognosis.¹²

According to the current guidelines, a tumor diameter greater than 2 cm serves as the cutoff point for lesions that are at a higher risk of metastasis.^22,23 More so, lesions larger than 2 cm have a mortality rate that is 19 times higher than that seen with lesions smaller than 2 cm.²³ The depth of the tumor is commonly described by its Breslow thickness, based on a millimeter measure of invasion, or by Clark level, which describes the level of tissue plane involved.²⁴ As the Breslow thickness describes the thickness of a tumor, it can be used to predict a patient’s prognosis.²⁴ A Breslow thickness of 2 mm or greater is associated with a significantly greater risk of local and regional recurrence compared with tumors less than 1 mm thick.²⁵

Once the tumor invades locally and is left untreated, it can spread to the underlying fascia, fat, bone, and nerves.²⁶ The involvement of nerves, or perineural invasion, occurs in 2% to 14% of tumors.²⁷ When this does occur, especially with larger-caliber nerves (>0.1 mm), it is often a poor prognostic factor with the risk of local recurrence and nodal metastases as high as 47% and 35%, respectively, following wide local excision.⁵

The anatomic site in which the cSCC develops itself plays an important prognostic factor in the associated risk of developing future tumors or metastases.²⁸ cSCC that develops on highly innervated areas, such as the nonglabrous lip or ears, have a higher rate of recurrence.²⁹

One of the most important prognostic factors is the presence of nodal metastases, which reflects the invasion from the primary tumor site to a regional lymph node.³⁰ The primary tumor cells can metastasize and spread to the bone, brain, liver, lungs, or other anatomical regions.³¹ For high-risk lesions, determining nodal metastasis is critical in staging as well as guiding treatment, which may warrant collecting lymph nodes surgically to be biopsied.³¹

Once the lesion data have been collected, including size, depth, nodal involvement, and metastases assessment, the tumor can be staged using a variety of classification schemes. The one commonly described by the American Joint Committee on Cancer is based on tumor burden (T), nodal status (N), and metastatic disease (M), referred to as the TNM grading system.³¹ This system, in turn, stratifies the risk of the tumor based on a variety of factors to help guide the clinician and patient toward appropriate treatment plans.³¹

Research is ongoing to find a significant pathway that drives cSCC tumorigenesis, as the Hedgehog signaling pathway does to BCC tumorgenesis.³² Whole-exome sequencing of cSCC revealed a set of oncogenic driver mutations that are commonly found in most cSCCs.³² The mutation load of cSCC demonstrates an average of 1 mutation per 30,000 base-pairs, which has posed challenges in the identification of key driver mutations.³² Notably, BCC differs from cSCC as BCC can arise de novo, whereas cSCC can develop from precursor lesions (ie, AK, BD).³² Mutations acquired in cSCC but not in precursor lesions, such as AK, could be the mutations that drive precursor lesions to become malignant.³²

From the available literature, mutations in driver and tumor suppressor genes have been associated with early events that cause DNA damage.³³ The most common of these events have been linked to chronic UV exposure, which results in characteristic C > T mutation at pyrimidines (COSMIC signature 7).³⁴ Inactivation of the tumor suppressor protein 53 (TP53) in epidermal keratinocytes and downregulation of NOTCH1 and NOTCH2 expression are early events in the majority of cSCCs, which results in the loss of tumor-suppressing properties of the NOTCH and TP53 pathways.³⁴

The TP53 gene’s role is to code the tumor suppressor p53 that is susceptible to mutations that can lead to a variety of cancers.³⁵ In the case of cSCC, if DNA becomes damaged from UV exposure, trauma, carcinogens, or any other factor, the transcriptional response of TP53 determines whether the cell is repairable or should undergo apoptosis.³⁶ This is a critical step in preventing tumor formation as TP53 function contains the damage and inhibits further spread.³⁶ Mutated TP53 is associated with over half of all known human cancers and, in the case of cSCC, has been considered the most commonly mutated gene.³⁷ When a TP53 mutation does occur, this causes the tumor cells to prevent apoptosis and propagate clonally to the detriment of nearby normal keratinocytes.⁵

RAS proteins (ie, HRAS, NRAS, KRAS) are known for their ability to regulate cell growth and proliferation.³⁸ They are small guanosine triphosphate proteins that activate multiple signalling pathways, which in turn activates proteins (eg, BRAF) downstream that ultimately express genes involved in cell survival, proliferation, and differentiation.³²RAS mutations are often activating, which can cause unintentional and overactive cell signalling downstream of incoming signals.³²RAS mutations can be found in approximately 20% of all tumors.³⁸ In a study by Li et al following the evaluation of 29 metastatic cSCC samples, most activating mutations affected KRAS and HRAS, resulting in the activation of the EGFR/BRAF/MEK/ERK pathway.^32,39

CDKN2A maps to chromosome 9 in a region that demonstrates a high frequency of loss of heterozygosity (LOH) and encodes for 2 cell-cycle regulatory proteins, p16^INK4a (p16) and p14^ARF (p14), which are associated with retinoblastoma and p53 pathways, respectively.^32,40 These proteins are responsible for regulating the progression of the cell cycle; thus, a mutation can result in uncontrolled cellular proliferation.⁴¹ AK can progress to cSCC when mutations, homozygous deletions, or LOH of CDKN2A occur.³² CDKN2A may have an important role in the formation of metastatic tumors, and across the published literature, the frequency of alterations ranges from 23% to 45%.³² 

The NOTCH signalling pathway functions in the cellular processes of proliferation, differentiation, and apoptosis.⁴²NOTCH1 (Truncated Allele of NOTCH [TAN-1]) and NOTCH2 are genes that are part of the NOTCH family of transmembrane receptors and are direct targets for the transcription factor p53.^32,43 The NOTCH signaling pathway is vital to epidermal development, growth, and keratinocyte differentiation; thus, any alterations to NOTCH activity could affect these processes.³² NOTCH1 is mostly expressed in the epidermis, and NOTCH2 is primarily found in the basal layer.³² In more than 75% of cSCC cases, a loss-of-function in NOTCH1 and NOTCH2 has been identified.⁴⁴ Although NOTCH signaling can be either tumor-promoting or -suppressing depending upon tissue/cell context, experimental studies of cSCC indicate that NOTCH signaling pathways are tumour-suppressive.^45,46 Thus, loss-of-function analyses using either dominant negative approaches or targeted deletion of NOTCH1 in keratinocytes results in keratinocyte hyperplasia, particularly in the basal layer, and ultimately formation of cSCC. In experimental animals, NOTCH1 and NOTCH2 function to suppress proliferation by regulating signaling through TP53, which impacts activity of cyclin-dependent kinase inhibitors (eg, CDKN1A), apoptosis mediators (eg, Caspase-3), and the WNT pathway.⁴⁷ NOTCH1 and NOTCH2 also regulate keratinocyte differentiation and polarization, and their loss results in aberrant localization and expression of differentiation markers, such as KERATIN-14 (K14) and integrins (INTGB1 and INTGB4).⁴⁸

The current standard of treatment involves surgical resection of the affected tissue and several millimeters of the surrounding tissue.³² The resection can be performed by standard excision, curettage and electrodesiccation, Mohs’ micrographic surgery or cryosurgery, and either chemotherapy or radiotherapy, depending on tumor characteristics.³² Each surgical modality carries an associated risk, issues with healing, and often leaves deformities.³² The systemic approach with chemotherapy does not target the specific area, thus weakening the person’s immune system and leaving them susceptible to further opportunistic disease.³² cSCC is radiosensitive; therefore, radiotherapy is recommended for locoregional disease and can also be used alongside chemotherapy and newer targeted therapies.⁴⁹ In the healing process following surgery, oxidative stress and the inflammation resulting from the procedure contribute to skin tumor promotion.³² Therefore, to increase survival while reducing cost and morbidity, novel therapeutic approaches are needed to provide alternative therapies for high-risk patients.³²

Recently, there has been a significant expansion in the currently available biological agents for the treatment of cSCC—for example, EGFR inhibitors.³² The overexpression of EGFR commonly activates in RAS signaling, which is why it is a promising target for molecular therapy.³² The overall response rates of EGFR inhibitors range from 20% to 40% and have shown to be even more efficacious when combined with chemotherapy.⁴⁹ Cetuximab is a chimeric IgG₁ monoclonal antibody that inhibits EGFR, and it has been approved by the FDA as an acceptable treatment for high-risk cSCC.^32,49 Cetuximab can be used on its own, or it can be used with other available therapies to enhance its efficacy.³² Clinical trials have shown cetuximab to produce positive outcomes for locally advanced or regional SCC; however, it has been shown to be less useful for metastatic sites.³² Tyrosine kinase inhibitors, such as imatinib and gefitinib, target the EGFR pathway, and clinical trials have yielded some antitumour activity in recurrent and metastatic lesions.³²

Introduced in the 1970s with various targets, immunotherapy is now used to target immune checkpoint inhibitors and is a relatively new approach to cancer therapy.⁵⁰ In cSCC, the use of this approach has gained popularity as the cells bearing somatic mutations caused by UV radiation can be targeted by the immune system.⁵¹ For cSCC and for most other cancers, the primary immune checkpoint that is targeted is programmed cell death-1 (PD-1), as it is found in both primary and metastatic tumors.⁵¹ Cemiplimab is an FDA-approved human IgG4 monoclonal antibody that targets PD-1, which is used primarily for patients who cannot have curative surgery or radiation therapy with locally advanced or metastatic cSCC.^32,51 Cemiplimab has shown promising results in aggressive cSCC but is not without adverse events, such as rash, fatigue, diarrhea, and mucositis, along with more serious immune-mediated systemic events, including pneumonitis, colitis, hepatitis, and nephritis.³² This has caused cemiplimab to be used with extreme caution, and the agent is not a viable option for immunocompromised individuals, who are among the those that at highest risk for developing cSCC.³²

Currently, there is no topical agent licensed for treating cSCC.³² However, several topical treatments are available for AK, such as cryotherapy and photodynamic therapy or topical chemotherapeutics, such as 5-fluorouracil (5-FU), which can, in turn, help prevent cSCC.⁵² Topical treatments for cSCC offer many benefits, such as allowing for higher drug levels at the tumor site with potentially less toxicity.⁵³ Several case reports and research articles are showing the promising off-licence use of topical imiquimod or 5-FU treatment, either alone or in combination.³² Therapy with 5-FU works by inducing apoptosis by binding to thymidylate synthase and thus inhibiting thymidine synthesis.⁵³ A case report describing the successful resolution of AK and biopsy-proven invasive cSCC using topical imiquimod, 5-FU, and tretinoin application, along with multiple sessions of cryotherapy, proved to be efficient in this particular case.³² However, future randomized-controlled clinical trials are warranted to draw definitive conclusions.³²

Hyaluronan (HA) has long been used in the skin care industry for many years as it was most known for its anti-aging properties.^54,55 However, HA has since been widely adopted in many medical specialties due to its role in tissue repair and regulating tissue injury.⁵⁶ HA has been used in treatment and management of several conditions, including lung fibrosis, asthma, pneumonia, hepatitis, liver fibrosis, cardiac disease, diabetes, and many other medical conditions.⁵⁶ Given the numerous benefits that HA offers in many areas of the body, further research has been conducted on its role in cancer treatment.

As not every patient is a good surgical candidate, there is a growing number of alternative treatments that can be used to treat cSCC.⁵⁷ Currently, 49% of the molecules approved to treat cancer were made from compounds that were derived from natural products.⁵⁷ HA is a ubiquitous member of the glycosaminoglycan group of polysaccharides that is commonly found in most mammalian tissues.⁵⁸ In humans, over 50% of body HA is found in the dermis and epidermis.⁵⁹ HA is composed of a repeating chain of 2 disaccharides, N-acetyl-d-glucosamine and d-glucuronic acid.⁶⁰ HA has many unique rheological, viscoelastic, and biological properties, which are primarily dependent on the size of the polymer.⁵⁹ As HA is negatively charged due to the repeating units of glucuronic acid, it is hydrophilic, which is a property that provides turgor, elasticity, moisture, and volume to the skin.⁶¹

HA polymers are commonly grouped as high-molecular-weight (HMWHA, >500 kDa), low-molecular-weight (LMWHA, 20-500 kDa), and oligosaccharide (<20 kDa) polymers.⁶² The HA polymer present in homeostatic tissues, including skin, is predominantly HMWHA. This size of HA polymer has anti-inflammatory and antiproliferative properties and has been shown to be tumor suppressive.^62–64 However, LMWHA is primarily produced by ROS/NOS, and hyaluronidases released in response to tissue stress are most often pro-inflammatory, encouraging proliferation and invasion.⁶⁵ LMWHA has been linked to tumor initiation and progression.^63,66

HA is produced by 3 highly homologous integral plasma membrane synthases (HAS1-3).⁶⁷ Although homologous, each HAS is on different chromosomes, and evidence to date suggests they have different properties and can be tissue- and cell-specific in their expression.⁵⁸ For example, HAS1 and HAS2 are predominantly responsible for producing large-sized HA polymers, compared with HAS3, which produces smaller-sized HA.⁶⁸ Polymer synthesis by HAS enzymes begins via the cytoplasmic domains, and the newly formed HA is extruded through plasma membrane pores into the extracellular matrix (ECM).⁶⁹

There is a balance between HA production and breakdown that varies by anatomical location.^70,71 As we get older, this balance becomes disrupted, and there is a higher amount of degradation compared with the production of HMWHA, which ultimately results in the appearance of “aging skin” and a loss of some of the HMWHA’s beneficial properties, including the tumor-suppressing abilities.^70,72,73 The enzymes all work by hydrolyzing the β (1-4) linkages between the 2 disaccharides to create shorter chains, which are more readily absorbed and processed for removal.⁷⁴ Native HA can be depolymerized by free radicals that are often produced during inflammation, trauma, and exposure to environmental carcinogens (ie, UV, hydrocarbons, burns).^74,75 HMWHA is primarily fragmented by HYAL2, which forms intermediate-sized HA fragments, which are further broken down into disaccharides and monosaccharides by HYAL1 and HYAL3 into lysosomes that are cleared by endocytosis and then further degraded to simple sugars by glycosidase.⁷⁴ The degradation of HA from HYAL enzymes, along with ROS and NOS, can cause a disturbance in the homeostatic environment.^64,75

Both HMWHA and LMWHA bind to various receptors to activate signaling cascades that impact inflammation and proliferation.⁶⁴ These include cluster of differentiation 44 (CD44), receptor for HA-mediated motility (RHAMM), toll-like receptor (TLR), and hyaluronan receptor for endocytosis (HARE).^64,74,76 CD44 is an essential component in maintaining the pericellular HA matrix surrounding keratinocytes, as well as HA endocytosis and cell-cell adhesions.^77,78 When LMWHA interacts with CD44, it results in ERK1/2 phosphorylation and facilitates the associated proinflammatory process.⁷⁹ However, when a HMWHA/CD44 interaction occurs, the HMWHA has multivalent sites for synchronous interactions with multiple receptors; this results in CD44 clustering on the cell surface with no ERK1/2 activation.⁷⁹

On the other hand, RHAMM, which is not typically expressed in the epidermis under normal homeostatic conditions, is expressed during tissue inflammation or repair.^79,80 When present, it is typically found in the deepest layer of the epidermis, the stratum basale.⁷⁹ RHAMM can be found on the cell surface, in the cytoplasm, in the nucleus, or in the ECM.⁶⁵ When RHAMM is overexpressed, it is commonly associated with tumorigenesis and malignant processes.⁷⁹ Like CD44, RHAMM is subject to alternative splicing; however, the mechanisms by which it binds to HA differs from CD44 as CD44 binds to HA via a link module, while RHAMM binds to HA via a cluster of positively charged amino acids.⁸¹ RHAMM preferentially binds to LMWHA and cooperates with CD44 in proinflammatory signaling.⁸¹

Research over the past decade investigating whether long-lived cancer-resistant species could yield any potential clues against skin carcinogenesis have highlighted naked mole rats (Heterocephalus glaber).⁷⁹ These studies have demonstrated resistance against cancer-induced tumorigenesis and other chronic diseases, such as neurodegeneration and cardiovascular disease.⁸² One of the proposed mechanisms associated with this tumor resistance has been the accumulation of HMWHA in the epidermis, acting by resisting growth through a CD44-dependent mechanism.⁸³ This accumulation has been attributed to an increased presence of HAS2, enhancing synthesis and decreased HYAL expression, thus minimizing degradation.⁸⁴

More recently, in vivo studies looking into this pattern of increased HMWHA and decreased HYAL found benefits in breast cancer.⁷⁹ The overexpression of HAS2 and exogenous treatment of HMWHA increased cytoplasmic cytochrome C expression, caspase activity, ROS generation, and apoptosis in breast cancer cells in naked mole rats.⁷⁹ CD44 expression is enhanced in 2D, 3D, and in vivo models during exogenous HMWHA treatment.⁷⁹ Researchers were able to conclude that in naked mole rats, the CD44/HMWHA interaction helps to facilitate cancer cell apoptosis and restricts cell cycle progression as a mechanism of cancer resistance.⁷⁹

Oral SCC is typically the result of smoking or alcohol use but can also be caused by molecular alterations in certain oncogenes.⁸⁵ The oral mucosa is normally a form of stratified epithelium similar to that found in the epidermis.⁸⁶ Increased HA is associated with tumor growth and metastasis such that, in dysplastic epithelium, HA staining can be found in the most superficial layer, along with multiple localized areas with reduced intracellular and irregular signaling for HA.^85,86 More specifically, increased RHAMM is found in invasive tumors and thus should be explored as a future target for oral SCC.⁸⁵ Additionally, when the HA-CD44 signaling pathway is inhibited, it can result in reduced tumor cell migration and reduced tumor growth.⁸⁷

More recent research between HA and cutaneous BCC has shed further light on the importance of HA in the cutaneous carcinogenesis process.^74,79 A patented (WO2011/140630A1) HMWHA linked to phosphatidylethanolamine (HA-PE) was developed by Dr Turley’s lab at the University of Western Ontario.^74,79 The proprietary formulation has shown promising results in animal models.^74,79 Symonette et al found that the use of HA-PE increased the formation of pericellular coats, increased epidermal thickness, and reduced inflammation.⁷⁴ The addition of phosphatidylethanolamine helped the epidermis absorb and retain the HMWHA formulation.⁷⁴ A study by Liu et al, using a Ptch^+/LacZ/Hr^-/- mouse model to assess the effect of HA-PE on BCC, found that the HA-PE inhibited the tumorigenic potential of mutations by restricting proliferation and promoting apoptosis; this resulted in an overall reduction of cancer-initiating cells via apoptosis, all while maintaining a homeostatic environment.⁷⁹ The study suggested HA-PE as a potential prophylactic agent for BCC and, possibly, other cancers known to be mediated by a similar pathway.⁷⁹ Another study by Cousteils et al used a model to assess the effect of HA-PE on UVB-induced Hr-/-Ptch+/- mice developing BCC.⁸⁸ Researchers found increased inflammation in the HA-PE group, possibly from the breakdown of HMWHA to LMWHA fragments by UV radiation, ROS, or enzymatic activity.^65,79 More importantly, there was a significant reduction (~50%) in BCC in the HA-PE group compared with the control.⁸⁸ These studies provide insight into HMWHA being a potential target for treatment as well as for the prevention of NMSC.