The neurogenic bladder: medical treatment

Neurogenic bladder sphincter dysfunction (NBSD) can cause severe and irreversible renal damage and bladder-wall destruction years before incontinence becomes an issue. Therefore, the first step in adequate management is to recognize early the bladder at risk for upper- and lower-tract deterioration and to start adequate medical treatment proactively. Clean intermittent catheterization combined with anticholinergics (oral or intravesical) is the standard therapy for NBSD. Early institution of such treatment can prevent both renal damage and secondary bladder-wall changes, thereby potentially improving long-term outcomes. In children with severe side effects or with insufficient suppression of detrusor overactivity despite maximal dosage of oral oxybutynin, intravesical instillation is an effective alternative. Intravesical instillation eliminates systemic side effects by reducing the first-pass metabolism and, compared with oral oxybutynin, intravesical oxybutynin is a more potent and long-acting detrusor suppressor. There is growing evidence that with early adequate treatment, kidneys are saved and normal bladder growth can be achieved in children so they will no longer need surgical bladder augmentation to achieve safe urinary continence in adolescence and adulthood.
Introduction
Neurogenic bladder sphincter dysfunction (NBSD) can develop as a result of a lesion at any level in the nervous system, including the cerebral cortex, spinal cord, or peripheral nervous system. Neurologic conditions in children leading to neurogenic bladder dysfunction are predominantly congenital neural tube defects (including myelomeningocele, lipomeningocele, sacral agenesis, and occult lesions causing tethered cord). Acquired causes such as spinal cord tumors or trauma or sequelae of transverse myelitis are less frequent. Whereas from an etiologic standpoint neurogenic bladder dysfunction is a heterogeneous group, medical management will be similar irrespective of the underlying cause. The vast majority of knowledge about NBSD management comes from long-term experience with myelomeningocele (MMC), the most common neural tube defect.
Following the institution of a general treatment policy with advances in neurosurgical and orthopedic treatments in previous decades, governing the associated NBSD has become crucial for improving quality of life and life expectancy in children with neural tube defects. In MMC patients, disordered innervation of the detrusor musculature and external sphincter adversely affects bladder function, which if untreated not only leads to incontinence but also will cause secondary damage and dysfunction of both the upper and lower urinary tracts. Key elements in optimal NBSD management are early diagnosis (including NBSD typology) and early (presymptomatic) institution of adequate medical treatment. There is indeed growing evidence that management decisions made during infancy, which prevent both renal damage and secondary bladder-wall changes, potentially impact long-term outcomes for renal function and safe urinary continence.
After describing the pathophysiology of NBSD and its possible consequences, this paper focuses on diagnosis (including early identification of patients at risk), treatment goals, treatment tools, and practical management of NBSD.
Historical evolution
The management of NBSD in children has undergone major changes over the years. A first milestone was the introduction of clean intermittent catheterization (CIC) in 1972 [1]. CIC (combined with anticholinergics if required) has made “conservative” (medical) management a successful treatment option, with a good outcome for quality of life and kidney protection. Further important breakthroughs were the wider application of urodynamic testing in the evaluation of infants and young children with suspected NBSD [2–4] and the better pathophysiological understanding of the natural history of NBSD in patients with spina bifida. In spina bifida, the natural history of the urinary tract in untreated NBSD is one of progressive deterioration by the age of 3 years in up to 58% of patients [5]. Several reports have shown this deterioration to be directly related to increased intravesical pressure. In 1981, the bladder pressure at which urethral leakage occurred was found to be a useful predictor of unsafe bladder function [2]. The leak-point pressure, as it is now commonly referred to, has become accepted as one of the urodynamic parameters that allows clinicians to differentiate patients with relatively low or high risk for subsequent upper urinary tract deterioration. In 1984, detrusor external sphincter dyssynergia (DSD) was identified as an important factor leading to functional obstruction, and intravesical pressure was recognized as the pathophysiological mechanism of subsequent upper urinary tract deterioration [3]. Shortly thereafter, urodynamics in infants and children was shown to allow a functional classification of NBSD that correlated with clinical entities of incontinence and obstruction, an approach that has allowed the concept of individualized and presymptomatic therapy in high-risk patients [6].
Pathophysiology of the neurogenic bladder
Under normal conditions, the detrusor muscle, bladder neck, and striated external sphincter function as a synergistic unit for adequate storage and complete evacuation of urine. In healthy bladders, the change in bladder-filling pressure between empty and full is normally less than 10–15 cm H2O. Normal voiding pressures for males and females are from 50 to 80 cm H2O and from 40 to 65 cm H2O, respectively [7].
In patients with NBSD, disordered innervation of the detrusor musculature and external sphincter adversely affects bladder function. In recent years, it has become clear that children with this condition can be categorized into high- and low-risk groups for secondary damage from a neurogenic bladder based on intravesical pressure. When the detrusor (filling) pressure exceeds 40 cm H2O, glomerular filtration rate decreases and pyelocaliceal and ureteral drainage deteriorates, leading to obstructive hydronephrosis and/or vesicoureteral reflux [2, 8–10]. Even in the absence of reflux or upper urinary tract dilatation, high intravesical pressure can impair drainage of urine into the bladder. Any pathophysiologic process that causes either intermittent or continuous elevation of bladder pressure above 40 cm H2O places the child at risk for upper urinary tract dysfunction, urinary tract infections, and ultimately renal failure. Intermittent elevation of bladder pressure may occur from detrusor hypertonia, hyperreflexia, or both. Hyperreflexia may cause intermittent elevation of bladder pressure, especially if the external sphincter acts reflexively and tightens rather than relaxes in an attempt to prevent micturition [detrusor sphincter dyssynergia (DSD)]. Over a long period of time, hyperreflexia with pressures greater than 40 cm H2O may result in detrusor decompensation (areflexia from myogenic failure) or in detrusor hypertrophy with associated sacculations and subsequent diverticula formation. These pathophysiologic changes affect the elastic and vesicoelastic properties of the bladder and also result in mechanical ureterovesical junction obstruction. Continuous elevation of bladder pressure above 40 cm H2O may occur from a hypertonic detrusor or a hypertrophic small-capacity bladder secondary to outflow obstruction [11]. Bladder outlet obstruction is caused by DSD, or by fibrosis of the external urethral sphincter secondary to partial or complete denervation [3, 12, 13]. Bladder outlet obstruction will lead to elevated (pathologic) voiding pressures, which will contribute to either detrusor decompensation or hypertrophy. Finally, recurrent urinary tract infections due to bladder residue may aggravate damage to the neurogenic bladder through processes of transmural inflammation and fibrosis. Together with high intravesical pressures and/or vesicoureteral reflux, these lower urinary tract infections will lead to episodes of acute pyelonephritis and irreversible renal damage.
Management of the neurogenic bladder
General principles and treatment goals
The cornerstone of optimal NBSD management is early identification and characterization (typology) and the institution of proactive therapy. Crucial for long-term prognosis of patients with NBSD is the fact that the management must start before consequences of bladder dysfunction become apparent. From the outset, the goals of management are to prevent or minimize secondary damage to the upper urinary tracts and bladder from the primary neurogenic bladder dysfunction and to achieve safe social continence [14]. Thus, long before continence becomes an issue, starting from the first year of life, management is directed at creating a low-pressure reservoir and ensuring complete and safe bladder emptying.
Clean intermittent catheterization (CIC) or self-catheterization (CISC) in combination with anticholinergics (oxybutynin) is the standard therapy for children with neurogenic bladder dysfunction with detrusor hyperactivity and/or DSD [11, 15, 16]. This treatment is also feasible and effective in developing countries, where untreated neuropathic bladder is an important cause of preventable chronic renal failure [17, 18]. CIC enables complete bladder emptying and thus avoids bladder residues and consequent risks for infections. In the high-risk bladder with DSD, CIC also allows bladder emptying before the occurrence of otherwise “spontaneous” high-pressure voiding, which is known to be detrimental for kidney function and drainage. Oxybutynin, a bladder smooth-muscle relaxant, is used to improve bladder dynamics through suppression of detrusor hypertonicity and hyperreflexia. By doing so, oxybutynin eliminates (high-pressure) uninhibited detrusor contractions (and thus urinary leakage) and prevents high-pressure bladder storage (due to detrusor hypertonicity or low bladder compliance) and high-pressure emptying (in case of DSD).
Early management, including diagnosis and identification of the high-risk bladder
At birth, the majority of patients with neurogenic bladder has normal upper urinary tracts. Without proper management, urinary tract infections and elevated bladder pressures with secondary bladder-wall changes may cause upper urinary tract deterioration within 3 years in up to 58% [5]. One third of children who develop impaired kidney drainage do so within the first year of life [19]. The specific abnormalities vary considerably and are not predicted by the level of the spinal cord defect. Furthermore, the dysfunctional pattern may be dynamic, influenced by spinal cord surgery, tethering, and denervation. In the initial baseline evaluation, clinical observations must be completed with urinalysis (microscopy and culture), renal/bladder ultrasound, and cystourethrogram. These allow the experienced clinician to suspect the type of NBSD and to identify the high-risk subgroup. The next consideration is when to perform urodynamic studies.
Two different opinions exist in the literature on the use of urodynamic studies in the early evaluation and further follow-up. In one approach, urodynamic assessment has become an integral part of the initial evaluation and subsequent management, as it allows recognition of the different subtypes of NBSD (typology), proactive interventions, evaluation and guidance of therapy, and early detection of neurologic deterioration (such as symptomatic tethering of the spinal cord [20]). Advocates justify this approach of routine urodynamics to minimize the deleterious effects of high intravesical pressure by directly measuring it rather than indirectly suspecting it from the development of upper and lower urinary tract changes on serial radiologic imaging. Several studies have shown that early urodynamic evaluation of children with NBSD allows the prediction of which newborns are at risk for upper urinary tract deterioration. Urodynamic risk factors are low bladder compliance, intravesical pressure more than 40 cm H2O, and DSD [2, 3, 6, 21]. The alternative to urodynamic-based management is serial radiologic imaging to detect secondary evidence of high bladder pressure. Critics of newborn and early infancy urodynamics refer to a lack of standards for performance and interpretation, which might lead to unnecessary interventions [22, 23]. Those authors recommend careful history, physical examination, upper urinary tract imaging, and close follow-up during infancy and childhood, reserving urodynamic studies only for patients with evidence of urinary retention on physical examination, new-onset hydronephrosis or febrile urinary tract infection, or for evaluation to achieve continence. Proponents of this approach with selective urodynamics suggest that close monitoring with prompt intervention at first signs of deterioration is effective in protecting the upper urinary tracts (including preservation of nephrons and thus renal function in the long run). A remaining concern, however, could be that in this more expectant approach, high intravesical pressures may have already resulted in irreversible and avoidable damage to the bladder wall, resulting in small-capacity, low-compliance bladders later in life.
Although many questions regarding optimal evaluation and management remain unanswered, the consensus on the need of close surveillance, especially in the first years of life, plus the possibility that proactive treatment may be better for the bladder in the very long term, emphasize the need for an integrated approach in which clinical observations, serial imaging, and urodynamics are the basis for early adequate treatment.
Urodynamic studies: special considerations in children with NBSD
If properly performed, even with possible shortcomings in newborns and infancy, urodynamic studies allow direct diagnosis of NBSD and recognition of dysfunction subtypes. This functional classification allows adequate treatment for the different types and early proactive treatment for the bladder at risk [6].
It is important for the practitioner to understand the complexities involved in performing urodynamic studies in newborns, infants, and children. Urodynamic assessment can provide reproducible results in newborns and infants, but it requires attention to mechanical factors and filling rates. The younger the child, the higher the risk that mechanical factors (such as bladder-outlet obstruction by the catheter used for the investigation) may produce artificial information (elevated leak pressure or inability to void). It has also been shown that using a bladder infusion rate as close as possible to the natural filling rate is important for correct assessment of detrusor properties [24]. It is presumed that fast infusion rates overcome vesicoelastic detrusor properties, falsely indicating detrusor hypertonicity [11]. On the other hand, in children who have apparent low-pressure cystograms and who leak during filling (due to sphincter hypoactivity), detrusor hypertonia may be unrecognized [25]. In these children, it is important to perform a provocative study (including bladder neck occlusion with a balloon catheter) to identify unrecognized detrusor hyperactivity prior to bladder-neck surgery for treatment of incontinence. Electromyographic (EMG) evaluation of the external urethral sphincter is required to identify DSD. The use of concentric EMG needles is preferred, as it gives more reliable information than patch electrodes [11]. The combination of X-ray cystography with cystometrogram and sphincter EMG (video urodynamics) allows accurate evaluation of the link between intravesical pressure and vesicoureteral reflux and gives direct visual information of (dys)synergia between detrusor and sphincter mechanisms [26].
Clean intermittent catheterisation
In children with neurogenic bladder, CIC is the first-choice treatment to empty the bladder adequately (no residue, no infection) and safely (prior to high-pressure voiding), and it is a valuable tool for achieving continence. The wide variety of used materials and techniques for CIC does not seem to affect efficacy and safety as long as some basic principles are applied: proper education and training, clean and atraumatic application, and achievement of good patient compliance on a long-term basis. For education, training, and further guidance during follow-up, a dedicated continence nurse is invaluable. Patients and caregivers must understand what is wrong with the bladder/sphincter and why CIC is proposed for treatment, and they have to learn how to catheterize properly. CIC has been successfully used by parents even in newborns and infants, becoming a part of their everyday routine [27]. Some authors prefer early institution of CIC in all infants with NBSD, given the fact that by the age of 3 years, CIC will be required in all for achieving continence, and given the difficulties of starting CIC at toddler age [28]. Such early institution of CIC seems to improve family compliance and their ability to assist the child in coping with their disease and with CIC [29]. CISC can be successfully taught to boys and girls who are motivated and who have developed the required dexterity, mostly around the age of 6 years. The required frequency of catheterization depends on several factors: fluid intake, bladder capacity, and bladder filling/voiding pressures. In practice, it is recommended to catheterize six times a day in infants (linked with feeding time) and five times a day in school-aged children. Although reported incidences of CIC-related infection risks are variable, it is generally agreed that the risk is low as long as complete bladder emptying is achieved. Furthermore, reused supplies are not related to more urinary tract infections [30]. If symptomatic infections occur, these are mainly caused by incomplete bladder emptying, and CIC appliance by child or caregiver needs to be optimized. To prevent urethral strictures and false passage in boys, catheter lubrication and avoidance of forceful manipulation during catheter insertion are advocated. Nonreusable low-friction catheters are considered valuable in high-risk male patients with urethral false passage or very tense sphincters but are unnecessary in routine cases [31]. To maintain therapeutic compliance with CISC in adolescents, psychosocial support is often required. Neurogenic bowel dysfunction with constipation and fecal soiling can interfere with the institution of a successful CIC treatment. Retained stools may mechanically impair bladder filling, increase detrusor irritability, or contribute to urine retention. Stool incontinence increases the risk of bladder contamination and urinary tract infection. An effective bowel management program is therefore needed. Finally, given the high prevalence of latex allergy [32], in the spina bifida population, a strict latex-free approach is of extreme importance.
Pharmacologic treatment: anticholinergics
Of the anticholinergic agents available, oxybutynin hydrochloride is most commonly used, and long-term experience supports its safety also in newborns and infants [33]. Oxybutynin is a tertiary amine with a well-documented therapeutic effect on detrusor hyperactivity, and its effectiveness is attributed to a combination of anticholinergic (M3-selective receptor subtype antagonism), antispasmodic, local anesthetic and calcium-channel-blocking activity [34]. Several studies have shown its efficacy for decreasing the filling pressure, increasing the capacity of the neurogenic bladder, and preserving renal function [35–37]. The usual dose regimen of oral oxybutynin is 0.3–0.6 mg/kg per day in three doses.
In children with insufficient response or significant systemic side effects to oral oxybutynin, intravesical instillation of oxybutynin has been shown to be a highly efficacious, reliable, and well-tolerated therapy for children who would otherwise require surgical therapy [38–43]. Because a solution suitable for intravesical instillation was not available, crushed oxybutynin tablets were used in the earlier trials, with consequent problems of inconvenience and impracticability, and it was the belief of several authors that poor patient compliance could be resolved by an optimized drug preparation [40, 44]. It was subsequently shown that, indeed, eliminating the complex crushing preparation by child or parent makes intravesical oxybutynin therapy easy to use and acceptable for long-term therapy [41].
The mechanisms underlying the more potent and longer-acting detrusor-suppressive effects of intravesical oxybutyinin, as well as its better tolerability, have been investigated by several groups. It was demonstrated that a reduced first-pass metabolism of oxybutynin after intravesical instillation, resulting in a reduced generation of the N-desethyl metabolite, may explain the clinically relevant reduction of systemic side effects that characterizes intravesical compared with oral oxybutynin therapy [45]. In addition, these pharmacokinetic studies provided first evidence for a direct local rather than a systemic effect of intravesical oxybutynin on detrusor muscle [45]. Further evidence for a local effect of intravesically administered oxybutynin was provided by studies showing local (urothelial) accumulation, suppression of muscarinic receptor-mediated detrusor muscle contractions, and blocking of muscarinic receptors in bladder-afferent pathways [46, 47]. In most reports, intravesical oxybutynin is used in dosages between 0.3 and 0.6 mg/kg per day in two or three doses. Given its better tolerability compared with oral treatment, if required, intravesical dosages can be further increased up to doses of 0.9 mg/kg per day [43].
To date, the vast majority (∼ 90%) of patients can be treated successfully with the gold standard treatment of oxybutynin (oral or intravesical) and CIC. Other bladder-relaxant drugs include propiverine (10–15 mg b.i.d. or t.i.d., adult dose), trospium (20 mg b.i.d., adult dose), extended-release oxybutynin, and tolterodine (children 0.25–1 mg b.i.d., adults 1–2 mg b.i.d.). The current experience with compounds other than oxybutynin is still limited in children with neurogenic bladder [48, 49]. Botulinum A toxin injections into the detrusor muscle have been shown to be a potentially valuable approach in the neurogenic overactive bladder [50]. Repeated botulinum A toxin injections (as an alternative for or an additive to anticholinergics) could be considered to postpone or avoid surgical procedures in the small minority of children not responding to standard therapy with CIC and anticholinergics [51]. However, further investigations are required, given remaining concerns about costs and long-term efficacy and safety of prolonged botulinum A toxin administration. Although some authors have advocated alfa-receptor stimulation of the bladder neck, no validated medical treatment is available to enhance the bladder outlet.
Medical management of NBSD in clinical practice
Optimal management involves first, early diagnosis, including recognition of high-risk subtypes, and second, proactive institution of adequate treatment. Early proactive treatment of high-pressure dyssynergic lower urinary tracts is important in the long term, not only to preserve renal function [52] but also to prevent poor bladder compliance and the subsequent need for bladder augmentation [35]. Urodynamic assessment is used in newborns and infants to come to a functional classification of NBSD, allowing presymptomatic interventions in the high-risk groups and individualized treatment planning according to the type of dysfunction [6, 29, 53].
In clinical practice, four major subtypes can be used to describe NBSD (Fig. 1): sphincter overactivity combined with detrusor underactivity (type A) or overactivity (type B), and sphincter underactivity combined with detrusor underactivity (type C) or with detrusor overactivity (type D). The easiest type to treat is type A. This bladder type requires early treatment because of urine retention with high filling pressure and continuous leaking. Here, CIC alone is effective and sufficient and will make the bladder safe and infection free, and the patient will be dry in between (social continence). Good care to empty the bladder totally is most important to avoid bladder infections caused by residual urine. Dysfunctional type B will have high filling and high voiding pressures, being very unsafe from birth onward due to DSD. Here, the act of voiding has to be prevented. With oxybutynin, the overactive detrusor can be “pharmacologically converted” to an inactive reservoir (situation similar to type A), which has to be emptied with CIC. In type C, CIC reduces the degree of incontinence and offers much better control over urinary tract infections. To achieve continence, this type will at a later age need surgical intervention on the sphincter (e.g. sling operation). An important caveat here is that detrusor instability may emerge only after surgical improvement of outlet resistance. If this detrusor instability would remain unrecognized and untreated (with oxybutynin), bladder-outlet surgery would have converted a “wet but safe” into a “dry but unsafe” bladder. In the last dysfunctional subtype (type D), the bladder leaks due to detrusor instability and gradually becomes unsafe due to secondary bladder-wall changes with detrusor hypertrophy and loss of bladder compliance. Therefore, treatment consists of CIC combined with oxybutynin and, at a later age, bladder-outlet surgery.
Fig. 1Classification of the neurogenic bladder, with four subtypes (a–d) according to dysfunctional activities of sphincter and detrusor. For each subtype, clinical implications if untreated and principles of management are summarized
Once appropriate therapy has been initiated, adequate follow-up is required, with adjustments if needed (CIC frequency, medication dosing and administration route). Treatment efficacy can be assessed using clinical parameters (including CIC frequency and volume charts), urinalysis, renal and bladder ultrasound, X-ray cystography, and video urodynamics.
As long-term sequelae of insufficiently treated neurogenic bladders (renal scarring, noncompliant fibrotic bladder) already have their origin in the first years of life, the frequency of multidisciplinary follow-up visits must be age dependent (3× yearly up to age 3 years, 2× yearly in school-aged children, yearly in adults). Typically, urinalysis and ultrasound are performed at all visits, cystography to investigate unexpected upper urinary tract infections, and urodynamics periodically to verify that under treatment, the catheterized bladder volumes are age appropriate [54] and stored under safe pressure conditions (storage of expected bladder capacity at pressures below 30 cm H2O; see [55]).
With early instituted and optimal treatment, the large majority of patients can be adequately controlled without antireflux surgery or surgical bladder augmentation (Fig. 2). Augmentation cystoplasty is limited to a small group of patients in whom medical treatment fails (persistence of high filling pressures). In patients with insufficient sphincter activity, continence achievement will require bladder-outlet surgery in addition to medical treatment. In female wheelchair users, surgical intervention to provide a continent stoma will facilitate self-catheterization.
Fig. 2Suppression of detrusor hyperactivity with resolution of reflux by nonsurgical management. Illustrative patient with high-risk neurogenic bladder sphincter dysfunction (NBSD) (type B), urodynamically showing early unsafe filling pressures (A) with high-grade reflux (a) and urosepsis before treatment. Under clean intermittent catheterization (CIC) plus oxybutynin, the unsafe high-pressure bladder was converted into a safe low-pressure reservoir with good capacity and disappearance of the reflux at control cystography 3 months later (b). Severe systemic side effects, making continuation of oral oxybutynin impossible, disappeared after switching to intravesical oxybutynin. Further urodynamic evaluations (B: after first intravesical administration; C: after 4 months) documented adequate suppression of detrusor hyperactivity (modified from [40]). Long-term (currently 13 years) continuation of CIC and intravesical oxybutynin has resulted in a safe and adequate capacity bladder with social continence for the patient
Long-term outcome evaluation and need for life-long follow-up
Lifelong follow-up with further periodic investigations of upper urinary tract changes, renal function, and bladder status is extremely important. There are two reasons why long-term outcome evaluation in adulthood and life-long patient follow-up are indispensable. First, for the individual patient, therapy is a life-long requisite, and verifying preservation of the patient’s kidneys is only possible by repetitive assessment throughout adolescence and adulthood. Second, in general, detailed long-term follow-up data will show whether a treatment policy driven by long-term goals is sufficiently effective or requires further adaptations. The effectiveness of efforts preserving upper urinary tract function can only be judged by assessing the ultimate outcome once these patients have reached adolescence or adulthood [29]. In populations with NBSD, no consensus exists as to how renal status is ideally evaluated [56]. In clinical practice, upper urinary tract deterioration or protection is often monitored by radiographic images of hydronephrosis and vesicoureteral reflux. Modalities used to look at renal functions include nuclear imaging [dimercaptosuccinate acid (DMSA) renal scan], urinary concentrating ability, and glomerular filtration rate assessment. For the latter, creatinine (Cr) clearance can be used for patients who are socially continent; for others, inulin or Cr ethylenediaminetetraacetate (EDTA) clearance can be used. Which (combination) of these tests is best to evaluate renal function requires further investigation [56].
Conclusions
Medical management with CIC and anticholinergics is effective in preserving renal function and providing safe urinary continence in more than 90% of patients with a neurogenic bladder. Early diagnosis and treatment institution, long before continence becomes an issue at toddler age, can prevent both renal damage and secondary bladder-wall changes, thereby improving long-term outcomes. Compared with oral oxybutynin, intravesical oxybutynin has more potent and longer-acting detrusor suppressive effects with good tolerance and should be used prior to considering surgical therapies. Therapeutic goals should no longer be restricted to prevention of secondary damage to both upper and lower urinary tracts. Instead, our goal should be to achieve normal renal and bladder growth at safe bladder pressure, with appliance-free continence.

neurogenic bladder
intermittent catheterization
anticholinergics
urinary continence
urodynamics


Extracellular Proteolysis in Transgenic Mouse Models of Breast Cancer

Growth and invasion of breast cancer require extracellular proteolysis in order to physically restructure the tissue microenvironment of the mammary gland. This pathological tissue remodeling process depends on a collaboration of epithelial and stromal cells. In fact, the majority of extracellular proteases are provided by stromal cells rather than cancer cells. This distinct expression pattern is seen in human breast cancers and also in transgenic mouse models of breast cancer. The similar expression patterns suggest that transgenic mouse models are ideally suited to study the role of extracellular proteases in cancer progression. Here we give a status report on protease intervention studies in transgenic models. These studies demonstrate that proteases are involved in all stages of breast cancer progression from carcinogenesis to metastasis. Transgenic models are now beginning to provide vital mechanistic insight that will allow us to combat breast cancer invasion and metastasis with new protease-targeted drugs.
Introduction
Breast cancer progression is accompanied by increased expression of proteases that are capable of degrading the extracellular matrix (ECM) leading to cancer cell invasion and eventually to dissemination of cancer cells to other organs. The proteolytic capability of breast cancers is reminiscent of the inherent proteolytic capability of the healthy mammary gland. Extracellular proteases are necessary for the dynamic tissue remodeling that takes place during normal mammary gland development and pregnancy cycling (reviewed in [1]). During development, the invasive phenotype of terminal end buds is associated with a controlled proteolytic digestion of ECM in the fat pad, and side branching of the newly formed ducts requires a focal degradation of basement membrane. The proliferative response of the epithelium during pregnancy and lactation is accompanied by limited proteolytic activity. In contrast, the involution of the lactating gland to a resting gland after weaning restructures the entire gland and requires extensive degradation of ECM and basement membranes. Extracellular proteases are thus more abundant during involution than at any other stage during development and the pregnancy cycle. The similarity between the proteolytic repertoire of the normal gland and the proteolytic capability of mammary cancers is best illustrated by the fact that the cellular expression patterns of individual proteolytic components are very alike, especially when comparing involution and cancer [2]. In terms of extracellular proteolysis, breast cancer in many ways resembles a mammary gland in a state of continuous and uncontrolled tissue remodeling.
Matrix Metalloproteases and Plasminogen Activators
Two families of extracellular proteases have been extensively studied in relation to mammary gland morphogenesis and cancer. These are the matrix metalloproteases (MMPs) and the group of serine proteases that govern the activation of plasminogen (Plg). Figure 1 illustrates the connected extracellular proteolytic network of the MMPs and the Plg activation system (PA system).
Figure 1The PA and MMP systems in the mouse. Plasmin and MMPs are two major sources of extracellular proteolysis. Plg is converted to the active protease plasmin by one of three Plg activators, uPA, tPA, and pKal. The generation of plasmin is enhanced by the binding of uPA to the cell surface receptor uPAR, and by the binding of tPA to coagulated fibrin or to cell surface proteins (annexin II [86] and CKAP4 [87]). The conversion of pro-uPA to active uPA may be initiated by several proteases, depending on the tissue in question. Additional pro-uPA is then activated by plasmin in a positive feed-back loop. The PA system is tightly controlled by several serine protease inhibitors (serpins); the primary and most specific inhibitors are the Plg activator inhibitor PAI-1 that targets uPA and tPA, and the plasmin inhibitor α2-antiplasmin (α2AP). Major substrates of plasmin include fibrin, fibronectin, laminin, and other ECM proteins, latent TGF-β and other growth factors, and pro-MMPs. The 22 different mouse MMPs are all extracellular, and are either soluble or membrane-anchored. Like the serine proteases of the PA system, the MMPs are also synthesized as inactive proforms that require proteolytic activation. Ten of the 22 pro-MMPs, including the membrane-anchored MMPs, have a furin cleavage site and may be activated by furin-like proprotein convertases before they are secreted. The remaining pro-MMPs are activated extracellularly, typically by plasmin or by other MMPs. The MMP system is counterbalanced by a group of four inhibitors, TIMPs, that have varying specificities for individual MMPs. In addition, the membrane-anchored MMP inhibitor RECK regulates a subgroup of MMPs including MMPs-2, -9, and -14 [88]. An excess of plasmin or MMP activity may ultimately be cleared by α2-macroglobulin (α2M) which is a non-specific protease scavenger. The MMPs are collectively able to degrade any component of the ECM. Important substrates for the MMPs as a group include the native fibrillar collagens and all four major components of the basement membrane: collagen type IV, laminin, nidogen/entactin, and heparan sulfate proteoglycans.
The liver-derived proenzyme Plg is found in high concentration throughout the body. In contrast, expression of the specific urokinase- and tissue-type Plg activators (uPA and tPA) is induced locally and thus determines where and when Plg is converted to the active protease plasmin [3, 4]. Plasmin, activated by uPA, is important for a number of tissue remodeling processes that, in addition to pregnancy cycling of the mammary gland [5], include wound healing [6] and embryo implantation [7]. In contrast, tPA is acknowledged to be primarily involved in thrombolysis and neurobiology [3, 4] but is also active during wound healing [8]. A newly identified Plg activator, plasma kallikrein (pKal), is involved in adipocyte differentiation in the involuting mammary gland [9] and in wound healing [8].
The MMPs constitute a large family of 22 extracellular metalloproteinases in mice (24 in humans) [10, 11] (an updated list of human and mouse proteases is available at http://web.uniovi.es/degradome/). MMPs are essential for a number of physiological tissue remodeling processes during ontogenesis and adult life, including mammary gland development [12, 13], bone development [14], wound healing [15], and embryo implantation [7]. During mammary gland development MMP-2 is involved in ductal elongation, and MMPs-2 and -3 are involved in ductal branching [13]. MMP-3 is also rate limiting for adipocyte differentiation during post-lactational involution [16]. The ductal tree is eventually fully formed in mice that are deficient in MMP-2, -3 or several other MMPs, indicating that the remaining MMPs are able to replace their activity in a functional overlap.
Several members of the MMP and PA systems are involved in breast cancer progression. We will focus on the role played by these extracellular protease families during the various stages of breast cancer progression, from the initial hyperplastic lesion to disseminated disease, with particular emphasis on the insight gained from transgenic mouse models.
Mouse Models of Breast Cancer
The mouse mammary gland is ideally suited to study cancer progression for a number of reasons. First of all, there are many similarities between the human and mouse mammary glands (for detailed reviews see [17, 18]). Human and mouse mammary glands contain the same epithelial cell types: the ductal epithelial cells, luminal (milk secreting) epithelial cells, and the myoepithelial cells. They also contain adipose tissue and stroma, although the composition of the stroma differs between the human and mouse glands. One of the few differences between the species is that the epithelium in the mouse gland is surrounded by adipose tissue and a relatively sparse stroma, whereas the human mammary epithelium is encompassed by a specialized fibroblastoid stroma and is not in direct contact with adipocytes. The epithelial structure in the mature nulliparous mouse gland is also much less developed compared to the human gland and consists only of a branching but otherwise naked ductal tree. In contrast, the mature ductal tree in non-pregnant women contains groups of acini at the end of terminal ducts, named terminal ductal lobular units (TDLU). The mouse epithelium develops similar lobuloalveolar structures transiently during the estrus cycle in certain mouse strains [19] and always during early pregnancy, and in this way the resting human gland is structurally more similar to the pregnant mouse gland. In some transgenic breast cancer models, the earliest hyperplastic lesions may also resemble TDLU-like structures [20].
A second ideal feature of the mouse mammary gland is that it is readily accessible for monitoring tumor growth and for other experimental procedures. This has lead to the development of numerous transplanted models, chemically induced models and transgenic models. We will limit our discussion to the transgenic and chemically induced models since there are inherent problems of transplanted models that may introduce bias. Depending on the study design, these obstacles can include: (1) a species barrier between proteolytic components supplied by the transplant and the host (as documented for human and mouse uPA and its receptor uPAR [21]); and (2) a cellular inconsistency with respect to protease expression in the sense that the majority of proteolytic components are supplied by stromal cells in human breast cancers while epithelial-based cancer cell lines often express the very same proteolytic components [22]. This is presumably due to genetic alterations that have occurred during in vitro culture. Furthermore, (3) when gene-deficient mice are used as hosts for syngeneic transplantation, it is possible that a tumor-suppressive phenotype in the protease-deficient host is due to an immune response directed at the protease-producing transplant.
A third advantage of the mammary gland for cancer research is that there are more transgenic models of mammary cancer than for any other cancer form, primarily because relatively specific promoters are available for targeting oncogene expression to the mammary epithelium. The promoters most frequently used stem from the whey acidic protein (WAP) gene, and the mouse mammary tumor virus long terminal repeat (MMTV). The promoters are not equally active at all stages of mammary gland development and the pregnancy cycle [23]. The MMTV promoter is active during puberty but is greatly enhanced during pregnancy. The WAP promoter is primarily active from mid pregnancy and during lactation. Accordingly, cancer progression is accelerated by pregnancies in most transgenic models and some models only develop tumors after a number of pregnancies.
Finally, the tumor histopathology of several transgenic models is very similar to human breast cancers [17]. The histopathology seen in a single mouse model is, however, relatively uniform and often characteristic of the inducing oncogene, and in this way no single model replicates the variability of tumor phenotypes seen among different breast cancer patients [17, 24]. This drawback is to be expected, considering the fact that transgenic models rely on a well-defined initiating oncogene in mice with a homogenous genetic background (inbred strains). In contrast, human breast cancers arise from combinations of multiple mutations in women with heterogenous genetic backgrounds. A notable similarity between mouse and human tumors is the development of a reactive stroma and the appearance of extracellular proteolysis in the tumor microenvironment. There are, however, also significant drawbacks to the use of transgenic models, such as the fact that they express oncogenes in the entire mammary epithelium (or at least a large part of it) often giving rise to multiple tumors and in some models a general hyperplasia of the entire gland. By contrast, human tumors derive from a single site, therefore resulting in a single tumor in the breast.
A comprehensive list of transgenic breast cancer models is available at http://emice.nci.nih.gov/ and has recently been reviewed [23]. Here we will focus on models that have been studied in relation to extracellular proteolysis. Table 1 summarizes the studies that describe MMP or PA system intervention in transgenic and chemically induced breast cancer models. It is evident from Table 1 that the role of proteases has almost exclusively been studied in models, in which one of the four oncoproteins ErbB-2 (neu) [25], Ras [26], Wnt1 [27], or the polyomavirus middle T antigen (PymT) [28] are induced by the MMTV promoter. ErbB-2 is often overexpressed in human breast cancers. Ras and the downstream effector proteins of Wnt1 and PymT are also frequently activated in human breast cancer, although mutation/overexpression of Ras and Wnt1 are infrequent in breast cancer and there is no cellular counterpart of the PymT oncogene [23]. In all four transgenic models, tumor incidence is 100% or at least approaches 100% depending on the time allowed for observation [29–32]. Tumor incidence has only in a few cases [29, 32] been affected in protease intervention studies (Table 1). Tumor onset in the ErbB-2, Ras, and Wnt1 models can be accelerated by multiple pregnancies [25, 30, 32] and mice used for experiments are frequently mated continuously, at least in the Ras and Wnt1 models [30, 32]. The PymT model is exceptionally fast even in non-pregnant mice, where the average tumor latency is approximately 1.5 months [31] compared to 4–7 months for multiparous mice in the three other models [25, 30, 32]. In all four models the tumors metastasize primarily to the lungs although the models differ in aggressiveness. The PymT model is the most aggressive and the Wnt1 model the least aggressive [31, 32]. A notable exception to the use of transgenic models in protease studies is the chemically induced mammary tumors that are induced by orally administered 7,12-dimethylbenzanthracene (DMBA) with or without hormone supplementation [33, 34]. The DMBA model is primarily a carcinogenesis model, since the tumors generally do not progress to form metastases [33, 34].
Table 1PA system and MMP intervention studies in transgenic and chemically induced breast cancer models.Proteolytic interventionMouse br.ca. modelPhenotypic effect on mammary neoplasiaReferencetumor incidencetumor onsettumor growth ratefinal tumor sizelung metastasis incidencelung metastasis burdencomments and additional phenotypesPlg activation systemPlg nullMMTV-PymT↔↔ (↓ trend)↔↔↓↓reduced metastasis in 129:BSw:FVB mixed strain but not in FVB mice[44] and unpublished datauPA nullMMTV-PymT↔↔(↓ trend)↔↔↔↓+ reduced lymph node metastasis[31]PAI-1 nullMMTV-PymT↔↔↔↔↔↔ (↑ trend)tumor vascularization not affected[45]uPAR nullMMTV-PymT↔↔↔↔↔↔no phenotype in the FVB strainunpublished dataMMP protease family MMP overexpressionMMTV-MMP-3 transgenenoneyes**tumors develop in the CD1 mouse strain but not in mixed B6:DBA mice[33, 61], see comment in [90]WAP-MMP-3 transgenenoneyes**tumors develop in the CD1 mouse strain but not in FVB mice[62], see comment in [90]MMTV-MMP-7 transgenenonenohyperplasia in multiparous mice but no tumors develop spontaneously[29]MMTV-MMP-14 transgenenoneyestumors develop spontaneously[63]MMTV-MMP-3 transgeneDMBA gavage↓↓opposite expectation[33]MMTV-MMP-3 transgeneDMBA gavage + MMTV-TGFα↔↔[33]MMTV-MMP-7 transgeneMMTV-neu↑↑↔↔[29]MMTV-MMP-7 transgeneApcMin + ENU↔[91] MMP inhibitionMMP inhibitor-treatment (galardin/GM6001)MMTV-PymT↓↓↓unpublished dataMMP-2 nullMMTV-PymT↔↔↔↔↓↓Fingleton and Matrisian, pers. comm.MMP-7 nullMMTV-PymT↔↔↔↔↔↔Fingleton and Matrisian, pers. comm.MMP-7 nullApcMin + ENU↓transient reduction in number of mammary tumors[91]MMP-9 nullMMTV-PymT↔↔↔↔↓↓reduced metastasis in mixed B6:FVB mouse strain but not in FVB miceFingleton and Matrisian, pers. comm.MMP-9 nullMMTV-neu↑(meeting report [67])MMP-11 nullMMTV-ras↔↓↓↑bidirectional effect[30]MMP-11 nullDMBA gavage↓↓ovarian and mammary carcinomas[66]MMP-13 nullMMTV-PymT↔↔↔↔↔↔unpublished dataWAP-TIMP-1 transgeneWAP-MMP-3↓reduced incidence of hyperplasias, no tumors develop[62]Albumin-TIMP-1 transgeneDMBA gavage + MPA↓early hyperplasia not affected[34]Albumin-TIMP-1 transgeneMMTV-PymT↔↓↓↔[34]MMTV-TIMP-1 transgeneDMBA gavage + MPA↔[34]MMTV-TIMP-1 transgeneMMTV-PymT↔↔↔[34]MMTV-TIMP-2 transgeneMMTV-Wnt1↓↓↓↓reduced mean vessel size in tumors[32]TIMP-2 gene therapyMMTV-neu↓↓[92]Abbreviations: ApcMin multiple intestinal neoplasia model in mice with a mutated Apc allele; B6, C57BL/6J; BSw Black Swiss; DMBA 7,12-dimethylbenzanthracene; ENU N-ethyl-N-nitrosourea; MMP matrix metalloprotease; MMTV mouse mammary tumor virus long terminal repeat promoter; MPA medroxyprogesterone acetate; PAI-1 Plg activator inhibitor-1; Plg plasminogen; PymT polyomavirus middle T antigen; TIMP tissue inhibitor of metalloproteases; uPA urokinase plasminogen activator; uPAR uPA receptor; WAP whey acidic protein promoter.
A feature of any transgenic model that is particularly relevant to extracellular proteolysis is the transition from pre-invasive (carcinoma in situ, CIS) to invasive breast carcinoma. Most human pre-invasive lesions are ductal CIS (DCIS), and in these lesions the carcinoma cells accumulate in the ducts confined by the myoepithelial cell layer and the basement membrane. Proteolytic degradation of the basement membrane leads to depolarization of the myoepithelial cells and offers the carcinoma cells access to the surrounding periductal stroma [35, 36]. In some human breast carcinomas the tumor suppressive myoepithelium surrounding an emerging tumor can persist for a relatively long period of time. In the MMTV-PymT model the late stage tumors resemble human invasive ductal breast cancer, but the myoepithelium is lost very early in tumor progression and it is therefore a poor model of the CIS stage ([20] and unpublished data). The lack of an intact myoepithelium, as seen in the MMTV-PymT model, does not necessarily make the neoplastic cells invasive. These lesions fall in the broad category of mammary intraepithelial neoplasia (MIN), which includes CIS-like lesions but is used more generally in mouse tumors to describe pre-malignant epithelial cells that have significant nuclear atypia and are surrounded by an intact basement membrane [37]. A few transgenic breast cancer models have been described to develop CIS lesions with morphologic similarities to human DCIS and later progress into invasive breast cancer. These include the C3(1)-SV40-T prostate cancer model that also develops mammary lesions [38], the WAP-SV40-T model [39], and the MMTV-neu models [17]. However, the CIS stage has not been the focus of protease intervention studies so far.
Protease Expression in Transgenic Breast Cancer Models
Extracellular proteases are generally absent in resting mammary glands but are present as the gland undergoes the pregnancy cycle. The levels of a number of extracellular proteolytic components are particularly upregulated during post-lactational mammary gland involution. These include uPA, tPA, MMPs-2, -3, -9, -11, and their corresponding inhibitors PAI-1 and TIMP-1 [5, 40–42]. The upregulated proteases and protease inhibitors are presumably required for the orderly restructuring of the lactating gland to a virgin-like state (discussed later). During breast cancer development, a similar proteolytic program is activated in the diseased gland and extracellular proteases are abundant in invasive breast cancers of both humans and mice. Figure 2 gives a schematic comparison of the expression patterns of MMP and PA system components in human breast cancer and in the MMTV-PymT transgenic breast cancer model, which has been used to analyze the expression of several extracellular proteases. One study used laser microdissection to isolate cancer cells and stromal cells followed by quantitative RT-PCR to examine the expression level of MMPs and the PA system [43]. The study concluded that stromal tissue adjacent to cancer cells expresses higher levels of uPA, PAI-1 and MMPs-2, -3, -11, -13, and -14 than the cancer tissue. The predominantly stromal expression patterns of all these components in the MMTV-PymT model has been confirmed by in situ hybridization [31, 43–46]. Using immunohistochemistry, uPAR expression was found primarily in fibroblasts, endothelial cells, and in some macrophages in the MMTV-PymT model (unpublished data). In situ hybridization was used in another study on MMTV-PymT tumors to determine the expression pattern of four of the membrane-type MMPs: the transmembrane MMPs-14, -15, and -16 and the GPI-linked MMP-17 [46]. MMPs-14 and -16 were detected in the stroma whereas MMP-15 was the only protease found predominantly in the epithelium. MMP-17 expression was not observed in the MMTV-PymT tumors. The majority of these findings reflect the mRNA expression data for human breast cancer, since uPA [47], PAI-1 [48], and MMPs-2 [49], -3 [50], -11 [49], -13 [35], and -14 [51] all are found predominantly in the stroma (see suppl. data to review by Egeblad and Werb 2002 [10]). Immunoreactivity for uPAR is also primarily found in the stroma [52]. Figure 3 provides two examples of this close correlation between human and transgenic mouse tumors: MMP-13 is focally expressed in stromal cells in patient material [35] and in MMTV-PymT transgenic tumors [43] (Fig. 3c–d). Similarly, the primary uPA inhibitor PAI-1 is expressed in stromal cells in MMTV-PymT mice [45] and in breast cancer patients [48] (Fig. 3e–f). The PAI-1-producing stromal cells were identified as myofibroblasts in the human samples. It is of note that this cell type is uncommon in the normal mouse and human glands but is abundant in tumors from both transgenic mice (unpublished data) and breast cancers patients [53].
Figure 2Expression of MMPs and PA components in breast tumors in humans and in MMTV-PymT transgenic mice. MMPs and PA components are expressed by cancer cells or stromal cells in breast tumors or are present as ubiquitous plasma-derived proteins. The predominant source of each mRNA/protein is illustrated for human ductal breast cancer and for the MMTV-PymT model in mice. Only those proteases and related components that have been analyzed in the MMTV-PymT model are included in the comparison. Only mRNA analyses are included except for uPAR, which was analyzed by immunohistochemistry in both species. The “Not detected” category includes proteases that were sporadically expressed in the tumors. The data for the MMTV-PymT model: uPA [31]; PAI-1 [45]; uPAR, MMPs-7, -9, -10 (unpublished data); MMPs-2, -3, -11, -13, -14, uPA, PAI-1 [43]; MMPs-14, -15, -16, -17 [46]. The data for invasive ductal breast cancer: uPA [47]; PAI-1 [48]; uPAR [52]; MMP-2 [49]; MMP-3 [50], MMP-7 [89]; MMP-9 [72]; MMP-10 [89]; MMP-11 [49]; MMP-13 [35]; MMP-14 [51].Figure 3Examples of protease expression in breast cancer from transgenic mice and humans. Expression of MMP-13 during early invasion in mouse C3(1)-SV40-T breast carcinoma (a) and in human ductal carcinoma in situ (DCIS) (b). MMP-13 is focally expressed in periductal fibroblast-like cells (arrows in a, a’, and b’) in areas with early cancer cell invasion (inv) and is not expressed in adjacent non-invasive areas with in situ carcinoma (CIS). Expression of MMP-13 in invasive MMTV-PymT carcinoma (c) and in human invasive ductal carcinoma (IDC) of the breast (d). Focal expression of MMP-13 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both mouse and human breast cancers (arrows in c and d). Expression of PAI-1 in invasive MMTV-PymT carcinoma (e) and in human IDC of the breast (f). The expression of PAI-1 is seen in fibroblast-like cells located in the stroma surrounding invasive cancer cells in both the mouse and human breast cancers (arrows in e and f). All panels are in situ hybridization using 35S-labeled probes. Panels a, c–f are counterstained with haematoxylin and eosin, whereas b is combined with pan-cytokeratin immunohistochemistry and (b’) with CK14 immunohistochemistry for myoepithelial cells [35]. Bars: a,c, 200 μm; b, 100 μm; d–f, 150 μm.
Recently, the MMTV-Wnt1 model has also been used to study the expression levels of a number of MMPs during mammary tumor formation by RT-PCR and in situ hybridization [32]. MMPs-2, -3, -9, -13, and -14 displayed increased expression levels in hyperplastic glands and mammary tumors. MMPs-2, -3, and -9 were exclusively expressed in the stroma, while MMPs-13 and -14 were found predominantly in the cancer cells and to a lesser extent also in stromal cells. Again, most of these MMPs have been reported with similar expression patterns in human breast cancer, although MMPs-13 and -14 are predominantly stromal in ductal breast cancer [35, 51]. It is noteworthy that the MMTV-PymT and MMTV-Wnt1 models have broadly similar but not identical localization and expression of MMPs.
The initial expression of proteases in human breast cancer may be associated with the transition from DCIS to invasive ductal carcinoma. The transition to invasive cancer can be identified histopathologically in some DCIS lesions, particularly facilitated by immunohistochemical staining of myoepithelial cells and intraductal neoplastic cells. Expression studies of MMPs in human DCIS material have shown that MMP-13 is focally upregulated in microinvasive foci (Fig. 3b) in contrast to MMPs-2, -11, and -14, which are all more broadly expressed in the samples [35]. All four of these MMPs are expressed in periductal (myo)fibroblasts, indicating an intense stromal involvement during early invasion. Brummer et al. found that TIMP-1 is also upregulated in areas of early invasion [54]. Mimicking the human situation, the C3(1)-SV40-T model develops DCIS-like lesions, in which MMP-13 is upregulated specifically in those DCIS-like structures that show signs of invasion (Fig. 3a). In human samples, uPA and uPAR are also focally upregulated in areas of microinvasion together with MMP-13, suggesting that plasmin-directed proteolysis coincides with MMP activity in the transition to invasive cancer [36].
Thus, the extracellular proteases expressed in transgenic breast cancer models generally mirror those identified in human breast cancer both in terms of expression and localization. These findings suggest that transgenic models are useful tools for the study of protease involvement in breast cancer invasion and metastasis.
Functional Studies of Proteases in Breast Cancer Progression
Proteases in Post-lactational Mammary Gland Involution
Mammary gland involution following weaning can to some extent be regarded as a surrogate model for the role of proteases in breast cancer invasion due to the extensive tissue remodeling and extracellular proteolysis taking place. The first, reversible stage of involution is prematurely activated in Plg-deficient mice likely due to increased milk stasis during lactation [55]. However, the second, protease-dependent and irreversible stage of involution seems to be delayed in Plg-deficient mice [5]. Involution is eventually accomplished in Plg-deficient mice probably due to compensating action of other proteases including MMPs. In support of this hypothesis, implantation of TIMP-1 slow release pellets into involuting mammary glands delays alveolar regression [40], and likewise TIMP-3 null mice exhibit accelerated involution [56]. In contrast, transgenic overexpression of TIMP-1 in luminal cells did not provoke an involution phenotype [16, 57], which may be a cell-of-origin effect since endogenous TIMP-1 is produced by stromal cells during involution [40, 58]. Similarly, the accelerated involution observed in mice that overexpress MMP-3 in luminal cells [57, 59] could be an effect of overexpressing a stromal protein in luminal cells, and is not supported by evidence from MMP-3 null mice that do not show a change in mammary apoptosis [16, 57]. The fact that the PA system and the MMPs are required for the controlled tissue remodeling of the involuting mammary gland, suggests that it is the inherent proteolytic repertoire of the mammary gland that is reactivated in breast cancers.
Proteases in Hyperplasia and Carcinogenesis
The earliest stages of mammary carcinogenesis including the initial genetic alterations in the epithelial cells were previously considered to be independent of the surrounding stroma and extracellular proteolysis. It now seems that the structural integrity of the stroma is an essential factor in preventing carcinogenesis [60]. In fact, disruption of the ECM and cellular microenvironment caused by overexpression of MMPs-3, -7 or -14 in the mammary gland is sufficient to initiate mammary epithelial hyperplasia [29, 61–63]. Transgenic mice overexpressing MMP-3 or MMP-14 even develop tumors in the absence of a transgene-supplied oncogene, and a fraction of the tumors undergo malignant transformation [62, 63]. Similar transgenic overexpression of Plg activators has not been reported for the mammary gland, but a K14-uPA/uPAR double transgene elicits extensive skin hyperplasia [64]. The MMP transgenes all direct MMP expression to the mammary epithelium, using either MMTV or WAP promoters. Only MMP-7 is expressed by normal mammary epithelium [65], while MMPs-3 and -14 are expressed by the mammary stroma during development and in the mature gland [46, 61]. Thus, tumor growth and malignancy have only been reported in cases where extracellular proteases were expressed by cells that would not normally produce them. Nevertheless, ectopically expressed proteases act as independent tumor promoters, presumably by compromising the growth-inhibitory role of the stroma, and eventually lead to genetic instability as if they were conventional oncogenes.
In light of the independent tumorigenic ability of some MMP transgenes, it is not surprising that tumor onset in transgenic breast cancer models is promoted by increased MMP activity and delayed by decreased MMP activity in some studies (Table 1). Tumor onset is thus promoted by MMP-7 overexpression in the MMTV-neu model [29] and delayed by MMP-11 deficiency in the MMTV-ras model [30] or by TIMP-2 overexpression in the MMTV-Wnt1 model [32]. Similarly, the incidence of DMBA-induced mammary tumors is reduced in MMP-11-deficient [66] and in TIMP-1-overexpressing mice [34]. The results of two studies were inconsistent with the general finding: MMP-3 overexpression resulted in reduced rather than increased tumor incidence in the DMBA model [33], and MMP-9 deficiency led to increased tumorigenesis in the MMTV-neu model [67]. This suggests that some MMPs may have a protective function in mammary tumorigenesis, similar to the protective role of MMP-8 in chemical carcinogenesis of the skin [68].
In contrast to the documented influence of some of the MMPs on tumor initiation, the PA system (including genetic deficiency in Plg [44], uPA [31], PAI-1 [45], or uPAR [unpublished data]) does not affect tumor onset in the MMTV-PymT model. Neither does TIMP-1 overexpression [34] or genetic deficiencies of MMPs-2, -7, -9 (Fingleton and Matrisian, personal communication) or MMP-13 (unpublished data) in the MMTV-PymT model. At present it is difficult to determine whether these differences reflect the nature of the different proteases or perhaps that the very early tumor onset, which takes place during puberty in the MMTV-PymT model, is less sensitive to the proteolytic environment.
Proteases in Tumor Growth and Vascularization
Mammary hyperplasias and CIS lesions are generally supplied with oxygen and nutrients by the pre-existing vasculature of the mammary gland. Tumor expansion beyond a certain stage requires the formation of new blood vessels. Angiogenesis, the sprouting of new vessels from existing vasculature, depends on proteolytic dissection of the endothelial basement membrane and other ECM components. Endothelial cells penetrate a newly deposited fibrin-rich matrix and use it as a temporary scaffold for vessel structure [69]. Accordingly, the fibrinolytic capacity of the PA system is crucial for vascularization in certain experimental settings, and PAI-1 may play a dual role as a protease inhibitor and cell migration mediator [3]. In tumors from breast cancer patients, endothelial cells are a considerable source of uPA, PAI-1 and uPAR suggesting a possible role in vascularization [47, 48, 52]. This has not been experimentally confirmed in transgenic tumors thus far (Table 1). In the MMTV-PymT model, tumors grow equally fast in the genetic absence or presence of Plg [44], uPA [31], PAI-1 [45] or uPAR (unpublished data), indicating that vascularization is not rate limiting for tumor growth in these cases. It is likely that functional redundancy with other proteolytic components compensates for the absence of a single component in the intact tumor. Even in PAI-1-deficient tumors, vascular density was not significantly different from wild type tumors [45]. It is also possible that vascularization of spontaneous tumors can be partially accomplished by endothelial precursor cells in a process termed vasculogenesis that may be independent of proteases or depend on a different subset of proteases [11].
Deficiency of MMP-9 reduces tumor growth in transgenic models of skin or β-cell islet tumors, most likely due to a requirement for MMP-9 to release vascular endothelial growth factor (VEGF) from ECM deposits in order to activate the angiogenic response [70, 71]. In human breast tumors MMP-9 is expressed by a subset of inflammatory cells and vascular pericytes, which could suggest a similar role of MMP-9 in breast cancer vascularization [72]. However, the evidence is less clear in transgenic breast cancer models (Table 1). Final tumor size in MMTV-PymT-transgenic mice is not significantly affected by single gene deficiencies of MMPs-2, -7, -9 (Fingleton and Matrisian, personal communication), or MMP-13 (unpublished data). Deficiency of a single MMP may be compensated by the presence of other MMPs. In contrast, simultaneous inhibition of a number of MMPs through a liver-specific TIMP-1 transgene significantly reduced final tumor size (by 42%) in MMTV-PymT mice [34]. Transgenic overexpression of TIMP-2 also caused a significant reduction in tumor growth rate and reduced the mean blood vessel size in MMTV-Wnt1 mammary tumors, providing direct evidence for an effect on vascularization [32]. MMP-11 may be important at this stage, since MMP-11-deficient mice develop significantly smaller tumors in the DMBA [66] and MMTV-ras [30] models. Surprisingly, a broad spectrum MMP inhibitor (galardin) only resulted in a 50% reduction in final tumor size in the MMTV-PymT model (unpublished data). It is possible that some MMPs promote while others inhibit tumor growth and vascularization, and the net effect of inhibiting a whole range of MMPs is rather limited. A protective role against tumor growth and vascularization has been observed in non-mammary models for MMP-3 [73] and MMP-19 [74].
Proteases in Cancer Metastasis
In order to successfully form metastases, cancer cells must enter the lymphatic system or the bloodstream, survive in circulation and arrest in the vasculature, extravasate and finally be able to proliferate in the new tissue. There are several metastasizing transgenic models, but the majority of models metastasize rather infrequently and only when the primary tumors are quite large. In addition to the MMTV-PymT [28] and MMTV-neu [25] models, very few other breast cancer models metastasize sufficiently to allow rigorous quantification (reviewed in [75]). Metastasis can be greatly accelerated by combining e.g. an MMTV-neu model with a second mammary-specific transgene for VEGF or transforming growth factor-β1 (TGFβ1) [75–77], but these models require more elaborate breeding. Proteases are intimately associated with metastasis. A classic example is the osteolytic bone metastases that are so frequent in breast cancer patients. Breast cancer cells and the resident osteoclasts stimulate each other in a feed-back activation loop. Cancer cells stimulate MMP-dependent degradation of the collagen type I rich bone matrix by osteoclasts [78, 79]. As a consequence of bone lysis, latent growth factors are released from their deposits within the bone ECM, and are available to stimulate further cancer cell proliferation. Unfortunately, transgenic models generally metastasize to the lungs and in this way the models do not exactly mimic the organ preference of metastatic breast cancer in humans.
The PA system plays a unique role in metastasis at least in the MMTV-PymT model that metastasizes to the lungs [28] and lymph nodes [31] (Table 1). uPA [31], PAI-1 [45], and uPAR (unpublished data) are all expressed both in the primary tumor and in the lung metastases. Metastasis is significantly reduced in both Plg-deficient and uPA-deficient mice, while these gene deficiencies have no significant effect on the primary MMTV-PymT tumors [31, 44]. Absence of the specific uPA inhibitor PAI-1 has a slight metastasis-promoting effect, but the difference is not significant perhaps due to the presence of alternative uPA inhibitors [45]. Deficiency of uPAR does not significantly affect metastasis in the inbred FVB strain (unpublished data).
Plg and uPA may promote metastasis due to their fibrinolytic ability. Fibrin-deficient mice have not been studied in a transgenic model, but they are less susceptible to spontaneous metastasis from transplanted tumors [80] and lung colonization by tail vein-injected cells [81], suggesting that fibrin is important for sustained adhesion of circulating cancer cells in the target organ. Thus, metastasis may depend on an intact and balanced system of fibrin formation and fibrinolysis. Alternatively, Plg and uPA may promote metastasis by releasing growth factors from the ECM. Overexpression of relevant ECM-sequestered growth factors promotes breast cancer progression and metastasis in transgenic models [76, 77, 82, 83]. These growth factors include the possible plasmin substrates VEGF, TGFβ, and FGF (fibroblast growth factor), as well as the possible uPA substrate HGF (hepatocyte growth factor).
There is also considerable evidence linking the MMPs to invasion and metastasis (reviewed in [10, 84]). In MMTV-PymT mice treated with the broad spectrum MMP inhibitor galardin, the average lung metastasis volume is reduced by approximately 99%, in the context of primary tumors that are only reduced by half (unpublished data). Metastasis incidence is also reduced in MMTV-PymT mice that overexpress TIMP-1 through a liver-specific transgene, but the reduction is less pronounced and is absent with a mammary gland-specific TIMP-1 transgene [34]. It will take a considerable effort to define which MMPs are responsible for the metastasis reduction observed with these inhibitors. MMP-9 deficiency results in reduced metastasis in the MMTV-PymT model, although this phenotype was mouse strain dependent (Fingleton and Matrisian, personal communication). Deficiency of MMP-2, which is abundantly expressed in the stroma of MMTV-PymT tumors [43], also results in reduced metastasis in the same model (Fingleton and Matrisian, personal communication). MMPs-2 and -9 have previously been associated with invasion and metastasis in a number of studies [84]. In contrast, there is no effect on metastasis in MMTV-PymT mice that are deficient in either MMP-7 (Fingleton and Matrisian, personal communication) or MMP-13 (unpublished data). Similarly, overexpression of MMP-7 in mammary glands has no effect on lung metastasis in the MMTV-neu model [29].
MMP-11 may play an unexpected role in actually preventing metastasis. In the MMTV-ras model, MMP-11 deficiency results in a higher number of metastases despite a lower number and volume of primary tumors [30]. Apparently, the transition to invasive carcinoma is greatly accelerated in the MMP-11 null mice, indicative of a protective function of MMP-11. A similar protective role against metastasis has been reported in non-mammary models for MMP-3 [73] and MMP-12 [85]. Thus, while the general MMP inhibitor galardin has a large inhibitory effect on metastasis, several single MMP knock-out studies report no effect or even the opposite effect on metastasis in breast cancer models. This demonstrates the potential of MMP inhibitors in preventing spontaneous metastasis but also underlines the necessity of evaluating a second generation of MMP inhibitors for clinical trials that are more specific than previous compounds. Transgenic models with a well-characterized involvement of MMPs in metastasis could be a useful tool in testing such compounds.
Conclusion and Future Perspectives
Transgenic breast cancer models are invaluable research tools because they recapitulate the entire process from the initial genetic events in normal cells to metastatic disease. Furthermore, the very similar expression patterns of extracellular proteases in human and mouse breast cancer samples suggest that transgenic mouse models are ideally suited to study the role of proteases in carcinogenesis and the progression to invasion and metastasis.
We have only begun to examine gene deficiencies and transgenic overexpression of the various PA and MMP components in transgenic models. The studies that have been performed so far suggest that no single MMP or PA system component is likely to be all-important for any stage in tumor progression. Practically all of the protease intervention studies report partial phenotypes such as delayed carcinogenesis, slower tumor growth or reduced metastasis, if they report any phenotype at all. The absence of any absolute phenotypes, such as prevention of metastasis, suggests that many of the proteases have mutually overlapping functions. The challenge for developing novel therapeutics in the future will be to identify those proteases that can be targeted simultaneously to obtain a synergistic tumor-suppressive effect.
Another area that has so far only been thoroughly examined in one or two transgenic models is the expression patterns of proteases. Using protease array data, it may be possible to draw some more detailed parallels between individual mouse models and certain histopathological subtypes of human breast cancers. It is very possible that the collective of transgenic breast cancer models available, rather than any single transgenic, will be needed to adequately model the heterogenous human disease. Another open question is whether certain proteases or inhibitors are up- or downregulated in response to certain gene deficiencies. This may provide vital information on functional overlap between individual proteases.
Transgenic breast cancer mice may prove to be very reliable models not only for basic research but also for testing experimental anti-proteolytic therapeutics, due to the high degree of similarity with human breast cancer. However, the transgenic models also present some significant challenges. A key issue is that the experimental drug has to cross-react with the mouse version of the protease. Therefore, it may be necessary in some cases to develop two drugs in parallel, one for each species. Due to the extracellular nature of the PA and MMP systems, these represent excellent targets for therapeutic monoclonal antibodies. An elegant method for targeting extracellular proteases in transgenic mice is the development of monoclonal anti-mouse antibodies by immunizing protease-deficient mice with the target protease. In conclusion, transgenic breast cancer models will further the identification of extracellular proteases critical to cancer invasion and metastasis, and aid the development of new agents against the spread of cancer.

plasminogen
upar
dcis
fibrinolysis
urokinase upa
matrix metalloprotease mmp