Branched peptides as novel tumor-targeting agents for bladder cancer

Keywords::

• bladder cancer
• branched peptides
• diagnosis
• intravesical chemotherapy
• selective drug delivery
• treatment

Bladder cancer (BC) ranks ninth in worldwide cancer incidence. It is the seventh most common cancer in men and the 17th most common cancer in women, with more than 330,000 new cases and more than 130,000 deaths each year [1]. Globally, the incidence of BC varies significantly, with Egypt, western Europe and North America having the highest incidence rates, and Asian countries the lowest rates [1]. There will be approximately over 70,000 new cases of BC and over 14,000 deaths in the USA in 2011, where BC remains the fifth leading new cancer diagnosis and is fourth among men [2]. According to the Italian Association of Medical Oncology–Italian Association of Cancer Registries data, in Italy, 24,000 men and 4000 women were diagnosed with BC in 2011, and it is estimated that over 30,000 and over 34,000 BC diagnoses will occur in 2020 and in 2030, respectively [3]. The main clinical impact of BC is related to recurrence and progression of disease. Patients with BC have the highest lifetime treatment costs per patient among all cancers, due to the numerous retreatments for recurrence requiring a further transurethral resection and for progression of the disease requiring a radical cystectomy [4]. Up to 80% of BCs are non-muscle-invasive BC (NMIBC); however, as many as 70% of these NMIBCs may recur and up to 25% of patients will progress to invasive disease [1,5]. NMIBC represents a heterogeneous disease where the risks of both recurrence and progression may be estimated for individual patients using scoring systems and risk tables. To predict separately the short- and long-term risks of both recurrence and progression in individual patients, a scoring system and risk tables were developed by the European Organisation for Research and Treatment of Cancer [6]. The stratification of patients into low-, intermediate- and high-risk groups separately for recurrence and progression is pivotal to recommending further treatments [4]. Patients diagnosed with BC may suffer several recurrences during their lives, and a significant percentage of these recurrences are probably undetected tumors at white light cystoscopy, which were not initially resected, rather than true recurrent new lesions [4,7]. Consequently, there is a great need for improving the diagnostic accuracy of our technologies.

Porphyrin-induced fluorescence cystoscopy (photodynamic diagnosis) that uses photoactive porphyrins, such as hexylaminolevulinate or 5-aminolevulinic acid, which accumulate preferentially in neoplastic tissue and emit red fluorescence under blue light (blue light cystoscopy), has been reported to improve the diagnostic accuracy of conventional cystoscopy for detecting bladder tumors, particularly carcinoma in situ. The additional detection rate of photodynamic diagnosis was 20% for all tumors and 23% for carcinoma in situ in a cumulative analysis of prospective trials [7].

Recurrence and progression can be significantly reduced using adjuvant chemotherapy (early immediate instillation or repeat instillation regimens with mitomycin C, epirubicin or doxorubicin) and bacillus Calmette–Guérin (BCG) immunotherapy.

In a recent study based on a long-term follow-up (mean: 6.5 years; range: 0.3–21.6 years), patients treated with BCG, 2–6 weeks after transurethral resection, followed with cystoscopy and urinary cytology for high-grade NMIBC, had progression in 30% of the cases, with a high proportion of these progression events occurring ≥3 years after BCG [8]. Indeed, according to the European Association of Urology guidelines, risk of progression in NMIBC ranges between 0.2 and 17% at 1 year and between 1 and 45% at 5 years, representing a highly probable life-threatening event [4]. This suggests the clear need for more effective adjuvant treatments, which are mandatory to reduce disease recurrence and progression.

In this direction, the selective targeting of tumor cells should be the goal of modern cancer diagnosis and therapy, aimed at overcoming the limits of current standard diagnostic tools and the nonspecific characteristics of most anticancer agents, maximizing the cytotoxic effect on the target tumor cells [9]. Peptide receptor tumor targeting strategy is particularly attractive, since it provides the possible tumor target and its candidate peptide targeting agent at the same time [9,10]. There are several advantages over the commonly used antibodies or small molecules: the smaller size and easier penetration into solid tumors; easy chemical synthesis; stability in harsh conditions, often necessary for the coupling to functional units; and chemico–physical properties that make them either more suitable for membrane tumor targets or for selective internalization by receptor-expressing cells [9,10]. However, the development of peptide drugs has always been limited by their extremely short half-life. Bracci et al. demonstrated that peptides synthesized in an oligo-branched form obtained on a lysine core result in molecules that can retain, or even increase, peptide biological activity through multivalent binding, and are very resistant to proteolysis, thus having a higher in vivo activity compared with their correspondent monomeric structure [10–13]. These findings open new perspectives for the use of peptides. A proof of concept has been obtained with peptides reproducing the sequence of the endogenous peptide neurotensin (NT) in its tetrameric form (NT4), whose receptors are overexpressed in several human malignancies. NT4 binds to cell membrane receptors more efficiently than the monomeric homologous sequence, and can be coupled to different effector units for either imaging or therapy of cancer cells [14]. Indeed, NT4 can efficiently discriminate between tumor and healthy tissue in human surgical samples from colon or pancreas adenocarcinoma, as recently reported in a large series of patients [15]. Furthermore, conjugation of cytotoxic drugs to NT4 provides an in vitro peptide receptor-mediated cytotoxic effect, increasing drug selectivity toward receptor-positive cells [16]. NT4 conjugated to methotrexate or 5-fluorodexoyuridine, which resulted in having the best in vitro cytotoxic activity, were tested in tumor xenografted mice models and showed to reduce tumor growth by 60% and 50%, respectively [16].

In addition, monastrol, combretastatin and chlorambucil were coupled to NT4 and tested on different cancer cell lines, resulting in an enhanced cytotoxic activity [16].

Moreover, drug-armed liposomes or nanoparticles decorated with NT4 are much more effective than nude liposomes for intracellular drug delivery in cancer cells [17]. The liposome–NT4 strategy allows the possibility of internalizing a greater load of therapeutic and diagnostic (theranostic) agents inside the cell membrane, and potentially overcoming cell resistance due to the lack of receptors on anaplastic cell clones [17]. Indeed, the switch to a receptor-selective drug internalization may have three very important consequences: it can dramatically reduce drug nonspecific cytoxicity; it increases in vivo activity of the drug; and it may induce reversal of innate cell resistances, when these are produced by a mechanism of cell internalization or export of drugs [14]. Branched peptides may increase the selectivity of small molecules towards tumor cells, thus acting as ‘Trojan horses’ that selectively transport chemotherapy drugs into tumor cells, since they have the ability of turning nonspecific cytotoxic drugs into tumor-selective agents, which presently have a limited use in oncology due to their low selectivity and high nonspecific toxicity [14]. On these bases, NT4 as a theranostic agent has a high potentiality and represents a very interesting field of investigation. The potential drawback is represented by the systemic administration route and the consequent determination of systemic bioavailability, metabolism and systemic toxicity, giving a limitation in the short-term translation into preclinical and clinical use for the diagnosis and treatment of several tumors. On the contrary, NT4 as a theranostic agent seems to be especially attractive in cases of BC, thanks to the endocavitary utilization and to its capacity to bind receptors overexpressed in BC cell lines, as already shown in our preliminary results [18]. Indeed, in the case of BC, the adoption of the endocavitary route can significantly facilitate the preclinical and clinical application. This could become a valid methodology both for diagnosis as a new photodynamic tool, adding a fluorophore to NT4, and for prevention of recurrence and progression of NMIBC. In our ongoing multidisciplinary research project, we expect to confirm our preliminary data obtained in vitro on NT4 and its capacity to discriminate between tumor and healthy tissue in surgical bladder samples, in a larger cohort of patients [18]. The following in vitro steps will involve new classes of receptors expressed in human cancers, other than NT, opening the way to the most varied applications both in the diagnosis and therapy. The last step will be the in vivo phase of the research, in which we expect to confirm our data already obtained on nude xenografted mice in colorectal and pancreas adenocarcinoma, to further evaluate diagnostic and cytotoxic capacity directly on human beings, exploiting, in the case of BC, the advantages of the minimally toxic endocavitary route of administration, making the translation into clinical use extremely promising. NT4 branched peptides seem to be promising theranostic molecules, which may allow a very precise cancer detection and selective therapy, by a simple switch of functional units.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.