It is the most frequently dysfunctional signaling in sporadic CRC. When Wnt ligand, a secreted glycoprotein, binds to its Frizzled (Fz) receptors, the multifunctional kinase GSK-3β is inactivated and β-catenin, that acts both as E-cadherin cell-cell adhesion protein and as a transcriptional activator, is stabilized, accumulated in the cytoplasm and finally translocated into the nucleus where it interacts with members of the lymphoid enhancer factor (LEF)/t-cell factor (TCF) and activates specific target genes. In the absence of Wnt-signal, casein kinase 1 (CK1) and the APC/Axin/GSK-3β-complex, target β-catenin for ubiquitination and proteasomal degradation by its phosphorylation, so preventing its nuclear translocation (17,19).

Wnt signaling contributes to tumour cell proliferation and inhibition of differentiation but it is also a critical mediator of endothelial function (20,21). Norrin, a non-Wnt ligand, binds selectively to Fz receptor subtype-4 (Fz4) and induces canonical Wnt signaling in conjunction with cell surface co-receptor LRP5 (9,10). Recently it has been demonstrate that Norrin is produced by human CRC, that it directly regulates endothelial cell proliferation and behavior, and that all of the critical components necessary to respond to Norrin signals are expressed by endothelial cells in the tumour microenvironment (22).

PI3K/AKT/PTEN pathway, often found dysfunctional in same sporadic and hereditary CRC, activates cell growth and inhibit apoptosis in response to several extracellular stimulations, such as growth factors, cytokines, hormones, heat and oxidative stress, hypoxia and hypoglycemia.

Binding of growth factor to its receptor induces self-phosphorylation and activation of receptor itself. Consequently, PI3K is recruited at the plasma membrane level and so activated. Activated PI3K converts phosphatidylinositol (4,5)-bisphosphate (PIP2) into phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and binding of PIP3 to AKT anchors it to the membrane allowing its phosphorylation and activation via phosphoinositide-dependent kinase-1 (PDK1) (23–25).

AKT acts in many cellular processes such as control of metabolism, translation, apoptosis and the cell cycle, by phosphorylating several target proteins, such as BAD (BCL-2 antagonist of cell death), caspase-9, mTOR (mammalian target of rapamycin), GSK3 and β-catenin (26–28). PTEN, a negative regulator of PI3K/AKT pathway, is a tumour suppressor gene, whose alterations are involved in several sporadic and hereditary CRC, that carries out its rule by dephosphorylating and downregulating PIP3 levels (29). Somatic mutations in this gene, such as small quantitative alterations of its expression, were found to be associated to different types of cancer (30). Furthermore, its germ-line mutations cause PTEN hamartoma tumors (PHTS) syndrome, a hereditary disorder predisposing patients to onset of multiple neoplasms (31–33). Recently it has been suggested that PTEN protein, as many others tumour suppressor genes, acts in a quantitative manner (34), and quantitative changes in its expression levels could have a role in phenotypic variability that PHTS patients have shown (35).

Ras/Raf signaling determines mitogen-activated protein kinase (MAPK) activation, a group of serine/threonine kinase proteins that mediate signal transduction from plasma membrane to the nucleus, in response to several extracellular stimulations, such as growth and mitogenic factors (36,37).

Ras genes (H, K and N) are localized on chromosome 12 and encode for small proteins with GTPase activity bound to the plasma membrane. Ras mutations usually activate Ras signaling by enhancing GTP levels and so mediating Raf proteins (A, b and c) phosphorylation and activation (38). Raf proteins, in turn, transduce signaling between MEK (1 and 2) and ERK (1 and 2) proteins, inducing transcription of genes involved in the cell cycle, and transcription regulation, such as Myc, cyclin-D/CDK (39,40).

NF-κB is a signaling pathway that takes part in cell proliferation and inflammation mechanisms. It consists of five subunits acting as transcription factors, RelA/p65, c-Rel, RelB, p50/NF-κB1 and p52/NF-κB2, that are able to dimerize and are sequestered in the cytoplasm by Iκb proteins. The IKK complex, consisting of two catalytic (IKKα and IKKβ) and one regulatory (IKKγ) subunits, represents the major regulator of this pathway. It acts by phosphorylating Iκb proteins targeting them for proteasomal degradation. Iκb degradation releases NF-κB proteins in the cytoplasm. Thus, they are free to translocate into the nucleus and activate transcription of specific genes (41,42).

All the molecular pathways cross-talk with each other and are regulated by one another. Interestingly, concerted regulation exists between NF-κB, Wnt and other adhesion proteins such as E-cadherin. E-cadherin directly binds to β-catenin and NF-κB sequestering them at the plasma membrane level and subtracting them from the nucleus. When the transcription of WNT5A is activated by NF-κB, WNT5A binds its specific Wnt receptor inducing transcription of target genes such as snail, CD44 and YAP1. Snail upregulation, in turn, represses E-cadherin expression releasing β-catenin and NF-κB, available to translocate into the nucleus (43,44).

GSK-3β is a protein that has a pivotal role in cross-talk between Wnt and NF-κB (45). In colon and pancreatic cancer cells, GSK-3β positively regulates NF-κB activity and its activation confers a selective growth advantage to these cells, therefore acting as a tumour promoter (46). The molecular mechanisms underlying GSK-3β/NF-κB interaction remain to be further investigated. More than 100 proteins, involved in a wide spectrum of cellular processes, are substrates of GSK-3β, of which β-catenin and NF-κB inhibitor iκb are the most well known. Genes upregulated by β-catenin/TCF/LEF and/or NF-κB include proto-oncogenes, such as c-Myc and cyclin-D1, and genes regulating cell invasion/migration, such as Snail, CD44 and MMP-7 (47).

Recently, it was suggested that EMT could be a common biological mechanism in cancers, representing a good target for therapeutic intervention. EMT consists in an essential phenotypic conversion of epithelial cells into cells with mesenchymal phenotype. It is a reversible process that often occurs during embryonic development and tissue remodeling and also plays a critical role in early event occurring in invasion and metastasis of many types of cancer, including CRC (48). EMT regulation is orchestrated by a group of transcription factors, including snail, slug, zeb1 and twist, but tumour microenvironment is also involved in this conversion through different signals, such as TGF-β, EGF, Wnt and Notch (49,50).

During EMT, epithelial cells lose E-cadherin expression, that specifically guarantees the epithelial phenotype, destroy their intercellular adhesion, acquire mesenchymal characteristics and increase migratory and invasive properties (51). Furthermore, EMt program induces stem cell specific gene expression, thus promoting self-renewal capability (52,53). Thus, understanding the specific molecular mechanisms sustaining EMT is of great relevance to block cancer progression.

TGF-β pathway represents the main signaling that regulates EMT. Binding of TGF-β to its receptor II (TGF-β-RII) promotes RI receptor recruitment on the complex at the membrane level. The activated complex induces Smad proteins phosphorylation, that, in turn, translocates into the nucleus, switching on transcription of specific genes, such as Snail, Slug, Twist and ZEB, all genes that are involved in EMT (54). We recently demonstrated that LiCl, a GSK-3β inhibitor, induces mesenchymal-to-epithelial transition (MET) in vitro, suggesting that LiCl and GSK-3β could represent, respectively, interesting drug and target for CRC therapy (55).

Clinical manifestations of CRC depend on the location of the lesion. Both right and left colon lesions occasionally cause hematochezia, but more often bleeding is occult, causing anemia and fatigue. Rectal lesions cause hematochezia, bleeding and tenesmus. Up to 30% of patients with colorectal carcinoma are primarily diagnosed in an acute stage with sub/obstructing symptoms (60,61). In 20–25% of patients with colonic cancer and in 18% of patients with rectal cancer, metastases are present at the time of the first diagnosis (62,63). Although liver represents the most common metastases localization in CRC patients, 2.1% of these patients show lung metastases (64), with frequency about three times higher for patients with rectal cancer than for patients with colon cancer.

Appropriate diagnosis and staging are crucial to ensure a correct treatment strategy. In the last 10 years the mortality rate of CRC has decreased by more than 20% due to the rising developments in diagnostic techniques and optimization of surgical, adjuvant and also palliative therapies (62). A complete colonoscopy up to the cecum, coupled with biopsy for histopathological examination, is considered the gold standard to diagnose colorectal lesions, in view of its high diagnostic performance (65,66). This procedure allows the tumour localization and possibly the endoscopic excision of polyps, so simultaneously representing a diagnostic and a therapeutic opportunity (67).

The best results are exhibited for lesions >6 mm, showing sensitivity and specificity of about 98 and 99%, respectively (68). However, a substantial proportion of patients will have an incomplete colonoscopy due to poor bowel preparation, poor patient tolerance, obstruction or other technical difficulties. In these cases, additional computed tomography-colonography (CT or CTC) can contribute to the CRC diagnosis (69,70), as a potential alternative to the endoscopy, especially in patients with stenotic tumour and/or when colonoscopy turn out to be incomplete or difficult. Furthermore, ct has been established as a highly sensitive and specific diagnostic modality for lesions >10 mm (71).

Despite such promising data, CTC does not offer the opportunity of taking biopsies or immediate polypectomy and the patient needs to return for a colonoscopy, in case of detected lesions. Tumours with distal extension to 15 cm (as measured by colonoscopy) from the anal margin are classified as rectal, while more proximal tumours are classified as colonic. Rectal digital examination can identify cancers up to 8 cm above the dentate line. Imaging plays a crucial role in the diagnosis, staging assessment for specific (e.g., neoadjuvant) therapy and follow up of patients with colon and rectal cancer with the main function of defining the locoregional extent, identifying synchronous lesions and distant metastases.

CRCs are classified according to local invasion depth (T stage), lymph node involvement (N stage) and presence of distant metastases (M stage) (72). These stages are combined into an overall stage definition, which provides the basis for therapeutic decisions. Specifically, classification according to tumour, node and metastases (TNM) and Union Internationale Contre le Cancer (UICC) stage offers valuable prognostic information and guides therapy decisions.

The most used imaging modalities for staging of CRC are chest/abdomen/pelvis CT (63,73,74) and magnetic resonance imaging (MRI). CT has a sensitivity of 74–84% and a specificity of 95–96% in detection of CRC liver metastases (64,74). MRI evaluates liver lesions <1 cm in size with a sensitivity of 80–88% and a specificity of 93–97% (75). However, ct has shown to be poor in identifying nodal disease (76,77). Its specificity for lymph node staging is 55%, while the sensitivity is 76% (64). Nodal size is too unreliable as a predictor for malignancy and should not be used as an absolute tool to define whether lymph nodes are involved or not. In preoperative imaging of rectal cancer, MRI is recommend for local staging (78,79) and whole body CT for detection of distant metastases (80–82).

MRI is able to accurately define the local extension of rectal tumour, the mesorectal fascia involvement, circumferential resection margin (CRM) (78) and the relationship of the tumour to the sphincter complex. It has a high accuracy in detecting extramural spread and in identifying tumours with good (<5 mm invasion) vs. poor (>5 mm invasion) prognostic features. Moreover, MRI represents the most accurate imaging tool for identifying nodal disease with a sensitivity of 85% and specificity of 97%, not by a size criteria but better with a definition of border irregularity and heterogeneity. MRI therefore is essential to accurately stage patients with rectal cancer in order to select the indication to a preoperative treatment or defining the extent of radical surgery.

Endorectal ultrasound (ERUS) is another well established modality for the evaluation of the integrity of the rectal wall layers. With accuracies for T staging ranging between 69 and 97%, endorectal ultrasonography is currently the most appropriate imaging modality for the assessment of T1 tumours. It may also detect regional adenopathy, with a lower accuracy to identify nodal involvement compared to both CT and MRI. As described by swartling et al (83), the combination of MRI with ultrasound improves diagnostic accuracy.

For the very early tumours (stage 1) and for the malignant polyps [Haggitt 1–3, T1 sm 1(−2?) N0], after adequate staging by ERUS, rectal MRI and CT scan, local excision could be considered, by means of traditional transanal procedure or by a video-assisted technique, both transanal endoscopic microsurgery (TEM) (99,100) or transanal minimally invasive surgery (TAMIS) (101,102).

Surgery is the mainstay of treatment for early rectal tumours: TME is the appropriate procedure, both performed laparoscopically or open. Minimally invasive approach is slow to become diffusely utilized due to the long learning curve of laparoscopic surgery, when compared to open surgery it has shown better short-term clinical outcomes and some evidence of comparable oncologic results are now arising. Several trials have compared the different surgical techniques in terms of oncological radicality and survival outcome.

The CLASICC trial comparing laparoscopic to open surgery and including rectal resections, showed an increased positivity of the CRM in the laparoscopic group, but no significant differences in terms of survival rates after 6 years of follow-up when compared to open surgery (103,104).

The COREAN trial (105) compared laparoscopy to open surgery after neoadjuvant chemoradiotherapy (CRT), showed no significant differences in terms of CRM positivity between 2 groups: 4.1% in the open group and 2.9% in the laparoscopic group.

The COLOR II study (106) showed a higher CRM positivity in the open surgery compared to laparoscopic group (22 vs. 9%, P=0.014), but no significant differences in terms of morbidity, mortality, and complication rates.

More studies are therefore needed to verify the superiority of one surgical technique over the others in terms primarily of oncological radicality and disease free survival.

Robotic surgery, finally, would seem to overcome the technical difficulties of laparoscopy, but there are still no reliable data demonstrating its superiority in terms of oncological results compared to open and laparoscopic surgery (29,30,107,108).

Robotic approach resulted in a lower percentage of conversion to open surgery (109,110) and a superiority of robotic rectal resection in recovery of urinary voiding and sexual function (111–113). Data from two multicentric studies comparing robotic to laparoscopic TME (ROLARR and COLRAR) are ongoing and results are awaited to better define the role of robotic rectal surgery (114).

Neoadjuvant therapy in [(C)RT] allows radical surgery with TME also in locally advanced rectal cancer. This approach has led to a reduction of recurrence rates from 30–40% of a few decades ago down to 5–10% or even lower figures. Adjuvant and neoadjuvant approach to locally advanced rectal cancer has been long debated: while a NIH Consensus Conference in the early 1990s stated that the most appropriate treatment was postoperative CRT (115,116), in Europe numerous trials supported RT before surgery with moderately high and never lower than 30 Gy doses. This approach has also led to a decrease of the local recurrence rate (117).

Neoadjuvant chemoradiation became standard practice after the publication of the results of the German trial (118). The choice between standard long course chemoradiation and short course radiation is still related to the 'tradition' of the group.

Several studies were performed, many are still in progress and many of them showed that both options have to be considered valid (118,119).

The stockholm III trial (120,121) regarding the fractionation of radiotherapy (RT) and timing of surgery for rectal cancer, has randomized patients to preoperative short-course RT with two different intervals to surgery: 5×5 Gy and surgery within 1 week (group 1) or after 4–8 weeks (group 2) or long-course RT 25×2 Gy and surgery after 4–8 weeks (group 3). These two different preoperative RT regimens has shown that patients of group 1 were associated with more postoperative complications than the other and that the experimental schedule (group 2) was shown to be feasible and as safe as the established treatments. However, results about local recurrences, will be available later in 2015.

Bujko et al (122) published long-term results of a randomized trial comparing preoperative short-course RT with preoperative conventionally fractionated chemoradiation for rectal cancer. Chemoradiation (50.4 Gy in 28 fractions of 1.8 gy, bolus 5-fluorouracil and leucovorin) and surgery 4–6 weeks later did not increase survival, local control or late toxicity compared with short-course RT alone.

Ngan et al (123) published the results of a randomized trial comparing short RT and CRT long-course having as end point the local recurrence in T3 rectal cancer. Two different treatments were offered: in one, patients underwent pelvic RT 5×5 Gy in 1 week, early surgery and six courses of adjuvant chemotherapy; in the other, long course RT 50.4 Gy, 1.8 Gy/fraction, in 5.5 weeks, with continuous infusional fluorouracil 225 mg/m² per day, surgery in 4–6 weeks, and four courses of chemotherapy were given. The final results showed that there were not significant differences between the two schedules in terms of local recurrence, except that long RT was more effective in reducing local recurrence only for distal tumours.

Siegel et al (124) published a randomised study comparing short RT plus TME-surgery within 5 days in the first group and long RT with continuous infusion 5-fluorouracil plus TME-surgery 4–6 weeks later in the second group. After surgery all patients had adjuvant chemotherapy for 12 weeks. No difference was shown in terms of local recurrence rate between the two groups.

5-fluorouracil (5-FU) in infusion is now the drug commonly used in neoadjuvant treatment. The oral capecitabine gives the same effects and is much easier to manage by both the oncologist and patient (125).

Combinations of 5-FU and other cytotoxic drugs such as oxaliplatin and irinotecan, and targeted drugs, have been extensively explored during the past decade. Multiple phase II studies in the so-called 'locally advanced rectal cancer' have claimed better results [more down-sizing, higher pathological complete rates (pCR)]. When cetuximab was added to CRT with capecitabine and neoadjuvant chemotherapy with capecitabine-oxaliplatin in a randomized phase II study, the primary endpoint, pCR rate, was not increased, but more radiological responses (89 vs. 72%, P=0.002) and improved OS (96 vs. 81% at 3 years, P=0.04) were seen in the KRAS wild-type population (n=90) (126).