[Development of Holistic Cancer Treatment Centering Cancer Patients - From the Standpoint of Hypoxia and Hedgehog Signaling].

Recently, hypoxia that is one of cancer microenvironments, takes much attention. Because circumstance that we usually perform experiment is 20% O2 condition, it is likely that different signaling pathways may be activated in vivo cancer. We focused Hedgehog(Hh)signaling as one of activated pathways under hypoxia. It has been shown that Hh signaling is activated under hypoxia, followed by inducing malignant phenotypes in pancreatic cancer. Therefore, Hh signaling inhibitor should elicit anti-tumor effect. However, if we consider "whole-person therapy" we should confirm how Hh signaling affects the function of immune cells. In the present study, we describe hypoxia/Hh signaling/functions of cancer cells and immune cells focusing our previous results.

Nuclear expression of Y-box binding protein-1 is associated with poor prognosis in patients with pancreatic cancer and its knockdown inhibits tumor growth and metastasis in mice tumor models.

The objective of this study was to examine the implication of Y-box-binding protein-1 (YB-1) for the aggressive phenotypes, prognosis and therapeutic target in pancreatic ductal adenocarcinoma (PDAC). YB-1 expression in PDAC, pancreatic intraepithelial neoplasia (PanIN) and normal pancreas specimens was evaluated by immunohistochemistry, and its correlation with clinicopathological features was assessed in patients with PDAC. The effects of YB-1 on proliferation, invasion and expressions of cell cycle-related proteins and matrix metalloproteinases (MMPs) were analyzed by WST-8, cell cycle and Matrigel invasion assays, Western blotting and quantitative RT-PCR in PDAC cells transfected with YB-1-siRNAs. To verify the significance of YB-1 for tumor progression in vivo, the growth and metastasis were monitored after intrasplenic implantation of ex vivo YB-1 siRNA-transfected PDAC cells, and YB-1-targeting antisense oligonucleotides were intravenously administered in nude mice harboring subcutaneous tumor. The intensity of YB-1 expression and positivity of nuclear YB-1 expression were higher in PDAC than PanIN and normal pancreatic tissues. Nuclear YB-1 expression was significantly associated with dedifferentiation, lymphatic/venous invasion and unfavorable prognosis. YB-1 knockdown inhibited cell proliferation via cell cycle arrest by S-phase kinase-associated protein 2 downregulation and consequent p27 accumulation, and decreased the invasion due to downregulated membranous-type 2 MMP expression in PDAC cells. Tumor growth and liver metastasis formation were significantly suppressed in nude mice after implantation of YB-1-silenced PDAC cells, and the YB-1 targeting antisense oligonucleotide significantly inhibited the growth of subcutaneous tumors. In conclusion, YB-1 may be involved in aggressive natures of PDAC and a promising therapeutic target.

Hedgehog signaling regulates PDL-1 expression in cancer cells to induce anti-tumor activity by activated lymphocytes.

We investigated whether hypoxia-induced activation of Hh signaling contributes to PDL-1 expression in cancer and whether it affects the anti-tumor function of activated lymphocytes. Hypoxia augmented PDL-1 expression and inhibition of Hh signaling reduced PDL-1 expression under hypoxia. When activated lymphocytes were cocultured with cancers treated with a Hh inhibitor, activated lymphocyte cell numbers increased under hypoxia. In contrast, this increase was abrogated when cancer cells were treated with a PDL-1 neutralizing antibody. These results suggest that Hh signaling is one of regulatory pathways of PDL-1 expression under hypoxia and that inhibiting Hh signaling may induce lymphocyte anti-tumor activity.

Reoxygenation from chronic hypoxia promotes metastatic processes in pancreatic cancer through the Hedgehog signaling.

Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly types of malignancies because of its high ability to metastasize. PDAC is thought to be under hypoxic condition. Therefore, to investigate the mechanism of metastatic processes, chronic-hypoxia-resistant PDAC cells were newly generated under hypoxic condition for 3-6 months and reoxygenation experiments were performed using these chronic-hypoxia-resistant PDAC cells in in vivo-mimicking conditions. Proliferation, invasiveness and tumorigenicity in PDAC cells were significantly increased by reoxygenation. A Hedgehog (Hh) signaling component, Gli1, was significantly increased by reoxygenation. Gli1 knockdown inhibited reoxygenation-induced increases in proliferation and tumorigenicity and decreased invasiveness through suppression of matrix metalloproteinase (MMP) 2 and MMP9. Moreover, inhibition of Sonic Hh and Smoothened abrogated reoxygenation induced increases in proliferation and invasiveness. These results suggest that metastatic processes in PDAC are induced through activation of the Hh signaling pathway. Therefore, the Hh signaling pathway may be a therapeutic target for refractory PDAC in metastatic processes induced by reoxygenation.

Hedgehog signaling pathway is a potential therapeutic target for gallbladder cancer.

Gallbladder cancer (GBC) is a particularly deadly type of cancer with a 5-year survival rate of only 10%. New effective therapeutic strategies are greatly needed. Recently, we have shown that Hedgehog (Hh) signaling is reactivated in various types of cancer and is a potential therapeutic target. However, little is known about the biological significance of Hh signaling in human GBC. In this study, we determined whether Hh signaling could be a therapeutic target in GBC. The Hh transcription factor Gli1 was detected in the nucleus of GBC cells but not in the nucleus of normal gallbladder cells. The expression levels of Sonic Hh (Shh) and Smoothened (Smo) in human GBC specimens (n = 37) were higher than those in normal gallbladder tissue. The addition of exogenous Shh ligand augmented the anchor-dependent and anchor-independent proliferation and invasiveness of GBC cells in vitro. In contrast, inhibiting the effector Smo decreased the anchor-dependent and anchor-independent proliferation. Furthermore, the suppression of Smo decreased GBC cell invasiveness through the inhibition of MMP-2 and MMP-9 expression and inhibited the epithelial-mesenchymal transition. In a xenograft model, tumor volume in Smo siRNA-transfected GBC cells was significantly lower than in control tumors. These results suggest that Hh signaling is elevated in GBC and may be involved in the acquisition of malignant phenotypes, and that Hh signaling may be a potential therapeutic target for GBC.

Hedgehog Gli3 activator signal augments tumorigenicity of colorectal cancer via upregulation of adherence-related genes.

Hedgehog signal is re-activated in several cancers. In this study, we examined the role of Gli3 on malignant phenotype of tumorigenicity for colorectal cancer and its relationship with p53, WNT and ERK/AKT signals. Gli3 expression was detected in HT29 and SW480 (p53-mutant) cells, but not in DLD-1 (p53-mutant) or HCT116 (p53-wild type) cells by reverse transcription-polymerase chain reaction and immunocytochemistry. Full-length Gli3 transfection increased anchor-independent growth for all cells regardless of p53 status, with upregulation of adhesion-related genes. Exogenous Sonic-Hedgehog increased activator-type of Gli3 and colony formation in Gli3-positive HT29 and SW480 cells. After implantation of Gli3-FL or mock-transfectant DLD-1 cells into SCID mice, tumor formation was highly observed in only Gli3-FL-transfectant group. In clinical specimens, Gli3 expression was detected in subsets of colorectal cancer and related with poorly-differentiated histological type, while Sonic-Hedgehog was present with high incidence. In conclusion, activator Gli3 signal augments tumorigenicity of colorectal cancer irrespective of p53 status.

Intratumoral FOXP3+VEGFR2+ regulatory T cells are predictive markers for recurrence and survival in patients with colorectal cancer.

Previously, we have shown that CD8(+)T/FOXP3(+) cell ratio but not FOXP3(+) cell number alone is an independent prognostic factor for colorectal cancer. In the present study, we evaluated whether the number of intratumoral FOXP3(+)VEGFR2(+) (itFOXP3(+)VEGFR2(+)) T cells alone could be a predictive factor for survival prognosis in patients with colorectal cancer. Distribution of regulatory T cells (Tregs) at tumor sites derived from 88 patients with primary colorectal cancer was fluorescence-immunohistochemically examined. Relatively low number of itFOXP3(+)VEGFR2(+) cells significantly correlated with better [corrected] disease-free survival (DS) and overall survival (OS); multivariate analysis indicated that number of itFOXP3(+)VEGFR2(+) cells is an independent predictive and prognostic factor of DS and OS while neither intratumoral FOXP3(+) cell number nor intratumoral FOXP3(+)VEGFR2(-) cell number alone showed significant correlation with DS or OS. These results suggest that FOXP3(+)VEGFR2(+) may be a better predictive Treg marker than FOXP3(+) alone for recurrence and survival in patients with colorectal cancer.

The Hedgehog inhibitor suppresses the function of monocyte-derived dendritic cells from patients with advanced cancer under hypoxia.

Immunotherapy using monocyte derived dendritic cells (Mo-DCs) from cancer patients has been developed; however, the Mo-DCs regularly studied have been derived from non-cancer bearing donors or mice, and evaluated in normoxic conditions. In the present study, we investigated the effects of Hedgehog (Hh) inhibitors which are being developed as molecular target drugs for cancer on the functions of Mo-DCs derived from patients with advanced cancer when cultured in a tumor-like hypoxic environment. Mo-DC induction, migration, chemotaxis, phagocytosis, maturation, IL-12 p40 or p70 secretion and the allogeneic lymphocyte stimulation activity of Mo-DCs from patients with advanced cancer were all significantly inhibited by the Hh inhibitor, cyclopamine under hypoxic conditions. Our results suggest that Hh signaling plays an important role in the maintenance and function of Mo-DCs derived from patients with advanced cancer when cultured under hypoxic conditions.

The Hedgehog inhibitor cyclopamine impairs the benefits of immunotherapy with activated T and NK lymphocytes derived from patients with advanced cancer.

Hedgehog (Hh) signaling is activated in various types of cancer and contributes to the progression, proliferation, and invasiveness of cancer cells. Many Hh inhibitors are undergoing clinical trial and show promise as anticancer drugs. Hh signaling is also induced in the activated T and NK (TNK) lymphocytes that are used in immunotherapy. Activated TNK lymphocyte therapy is anticipated to work well within a tumor's hypoxic environment. However, most studies on the immunobiological functions of activated TNK lymphocytes have been conducted on healthy donor samples, under normoxic conditions. In the present study, we evaluated the effects of Hh inhibition and oxygen concentrations on the function of activated TNK lymphocytes derived from patients with advanced cancer. Proliferation, migration, surface NKG2D expression, and cytotoxicity were all significantly inhibited, and IFN-γ secretion was significantly increased upon Hh inhibitor treatment of activated TNK lymphocytes under hypoxic conditions in vitro. Tumors from mice injected with cyclopamine-treated activated TNK lymphocytes showed a significant increase in tumor size and had fewer apoptotic cells compared with the tumors in mice injected with control activated TNK lymphocytes. These results suggest that Hh signaling plays a pivotal role in activated TNK lymphocyte cell function. Combination therapy using Hh inhibitors and activated TNK lymphocytes derived from patients with advanced cancer may not be advantageous.

Hedgehog inhibitor decreases chemosensitivity to 5-fluorouracil and gemcitabine under hypoxic conditions in pancreatic cancer.

Pancreatic cancer is one of the deadliest types of cancer. Previously, we showed that hypoxia increases invasiveness through upregulation of Smoothened (Smo) transcription in pancreatic ductal adenocarcinoma (PDAC) cells. Here, we first evaluated whether hypoxia-induced increase in Smo contributes to the proliferation of PDAC cells. We showed that Smo, but not Gli1, inhibition decreases proliferation significantly under hypoxic conditions. To further investigate the effects of Smo on PDAC growth, cell cycle analysis was carried out. Inhibition of Smo under hypoxia led to G(0) /G(1) arrest and decreased S phase. As 5-fluorouracil (5-FU) and gemcitabine, which are first-line drugs for pancreatic cancer, are sensitive to S phase, we then evaluated whether cyclopamine-induced decreased S phase under hypoxia affected the chemosensitivity of 5-FU and gemcitabine in PDAC cells. Cyclopamine treatment under hypoxia significantly decreased chemosensitivity to 5-FU and gemcitabine under hypoxia in both in vitro and in vivo models. In contrast, cis-diamminedichloroplatinum, which is cell cycle-independent, showed significant synergistic effects. These results suggest that hypoxia-induced increase of Smo directly contributes to the proliferation of PDAC cells through a hedgehog/Gli1-independent pathway, and that decreased S phase due to the use of Smo inhibitor under hypoxia leads to chemoresistance in S phase-sensitive anticancer drugs. Our results could be very important clinically because a clinical trial using Smo inhibitors and chemotherapy drugs will begin in the near future.

Inclusive estimation of complex antigen presentation functions of monocyte-derived dendritic cells differentiated under normoxia and hypoxia conditions.

Dendritic cells (DCs) generated from monocytes under 20% O2 are now used as therapeutic tools for cancer patients. However, the O2 concentration is between 3 and 0.5% in most tissues. We evaluated these complicated functions of DCs under oxygen tensions mimicking in vivo situations. Immature DCs (imDCs) were generated from monocytes using IL-4 and GM-CSF under normoxia (20% O2; N-imDCs) or hypoxia (1% O2; H-imDCs). Mature DCs (mDCs) were induced with LPS. DCs were further exposed to normoxia (N/N-DCs) or hypoxia (N/H-DCs and H/H-DCs) conditions. Using a 2-D culture system, H-DCs were smaller in size than N-DCs, and H/H-DCs exhibited higher allo-T cell stimulation ability than N/N-DCs and N/H-DCs. On the other hand, motility and phagocytic ability of H/H-DCs were significantly lower than those of N/H-DCs and N/N-DCs. In a 3-D culture system, however, maturation of H/H-imDCs and N/H-imDCs was suppressed compared with N/N-imDCs as a result of their decreased motility and phagocytosis. Interestingly, silencing of HIF-1α by RNA interference decreased CD83 expression without affecting any antigen presentation abilities except for the ability to stimulate the allo-T cell population. Our data could help our understanding of DCs, especially therapeutic DCs, in vivo.

Hedgehog signaling pathway as a therapeutic target in various types of cancer.

Hedgehog (Hh) signaling is an important factor in growth and patterning during embryonic development. A mutation in Patched, Smoothened or Gli1, which regulate the Hh signaling pathway, might lead to the onset of glioblastoma, basal cell carcinoma, medulloblastoma and rhabdomyosarcoma. Recently, Hh signaling has been reported to be activated in a ligand-dependent manner, contributing to carcinogenesis and cancer progression. Hedgehog signaling is reactivated in various types of cancer, and this contributes to cancer progression by facilitating proliferation, invasion and cell survival. Moreover, Hh signaling is associated with several other signaling pathways that contribute to cancer progression. These observations indicate that controlling Hh signaling might become a target for novel molecular targeting therapy.

Semi-quantitative evaluation of CD44(+) /CD24(-) tumor cell distribution in breast cancer tissue using a newly developed fluorescence immunohistochemical staining method.

CD44(+) /CD24(-) tumor cells are reported to contain cancer stem cells in breast cancer. The main purpose of the present study is to develop an immunohistofluorescence method that can quantitatively analyze CD44(+) /CD24(-) tumor cell distribution in breast cancer tissue and help better define the role of CD44(+) /CD24(-) tumor cells in breast cancer. The samples used were from 21 primary breast cancer patients who underwent neoadjuvant chemotherapy and 17 cases with sentinel lymph nodes that had lymph node micrometastasis. CD44(+) /CD24(-) tumor cells were distinguished at a single cell level using improved triple-staining immunohistofluorescence and a simulated laser capture microdissection method. The percentage of CD44(+) /CD24(-) cells significantly increased following neoadjuvant chemotherapy treatment (0.93% and 2.78%, before and after, respectively, P = 0.0043). The percentage of CD44(+) /CD24(-) cells was also significantly high in micrometastatic sentinel lymph nodes (0.49% and 1.91%, primary tumors and lymph nodes, respectively, P = 0.0246). The CD44(+) /CD24(-) tumor cell distribution was heterogeneous in both breast cancer tissue and lymph node metastasis. In a xenograft model using immunodeficient mice, the hedgehog signaling inhibitor cyclopamine repressed the tumorigenicity of CD44(+) /CD24(-) cells. Our results suggest that this semi-quantitative immunohistochemical analysis is valuable for detecting a small population of cells in cancer tissues and that the hedgehog signaling pathway inhibitor cyclopamine is useful for regulating the CD44(+) /CD24(-) tumor cells in breast cancer.

Hypoxia activates the hedgehog signaling pathway in a ligand-independent manner by upregulation of Smo transcription in pancreatic cancer.

The hedgehog (Hh) signaling pathway is activated in various types of cancer including pancreatic ductal adenocarcinoma. It has been shown that extremely low oxygen tension (below 1% O2) is found in tumor tissue including pancreatic ductal adenocarcinoma cells (PDAC) and increases the invasiveness of PDAC. To investigate the contribution of the Hh pathway to hypoxia-induced invasiveness, we examined how hypoxia affects Hh pathway activation and the invasiveness of PDAC. In the present study, three human PDAC lines were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. Hypoxia upregulated the transcription of Sonic hedgehog (Shh), Smoothened (Smo), Gli1 and matrix metalloproteinase9 (MMP9) and increased the invasiveness of PDAC. Significantly, neither the addition of recombinant Shh (rhShh) nor the silencing of Shh affected the transcription of these genes and the invasiveness of PDAC. On the other hand, silencing of Smo decreased the transcription of Gli1 and MMP9 and PDAC invasiveness. Silencing of Gli1 or MMP9 decreased PDAC invasiveness. These results suggest that hypoxia activates the Hh pathway of PDAC by increasing the transcription of Smo in a ligand-independent manner and increases PDAC invasiveness.

Hedgehog signaling pathway mediates the progression of non-invasive breast cancer to invasive breast cancer.

The purpose of this study is to clarify the contribution of the Hedgehog signaling pathway (Hh pathway) to the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). A total of 149 surgically resected mammary disease specimens and 12 sentinel lymph nodes with micro-metastasis (Ly-met) were studied. The degree of Hh pathway activation was estimated from the Gli1 nuclear staining ratio (%Gli1 nuclear translocation) in cancer cells. The invasiveness of breast cancer cells was determined using Matrigel assays. A serial increase of %Gli1 nuclear translocation to IDC from non-neoplastic diseases was confirmed. In tumor specimens, %Gli1 nuclear translocation correlated with the invasiveness of each type of mammary disease and also correlated with invasion-related histopathological parameters. The %Gli1 nuclear translocation in lymph nodes with micro-metastasis was similar to that in primary sites and higher than that in DCIS with microinvasion and DCIS. Blockade of the Hh pathway decreased the invasiveness of breast cancer cells. In IDC, %Gli1 nuclear translocation correlated with the expression of estrogen receptor-α. Estrogen increased %Gli1 nuclear translocation and the invasiveness of estrogen receptor-α-positive cells. The Hh pathway mediates progression from a non-invasive phenotype to an invasive phenotype and %Gli1 nuclear translocation may be useful as a predictive marker for evaluating the ability of invasiveness.

VEGFR2 is selectively expressed by FOXP3high CD4+ Treg.

CD25+ FOXP3+CD4+ T cells (Treg) have been considered to play an important role in immune tolerance against several tumor antigens. It has also been indicated that high-level expression of FOXP3 (FOXP3high) is sufficient to confer suppressive activity to normal non-Treg. Here, we showed for the first time that vascular endothelial growth factor receptor 2 (VEGFR2) is selectively expressed by FOXP3high but not FOXP3low Treg. Such VEGFR2+ Treg exist in several tissues including PBMC and malignant effusion-derived lymphocytes. In conclusion, VEGFR2 may be a novel target for controlling Treg with highly suppressive function.

Objective response with lapatinib in patients with meningitis carcinomatosa derived from HER2/HER1-negative breast cancer.

A 45-year-old woman with HER2(-)/HER1(-) breast cancer underwent radical mastectomy, followed by radiation and chemotherapy. However, her symptoms progressed rapidly owing to meningitis carcinomatosa and she was fitted with a urethral catheter. She also had difficulty in walking. However, immediately after treatment with lapatinib, her symptoms almost completely disappeared. The catheter was removed and she no longer needed a wheelchair. Unfortunately, after treatment was stopped, the bilateral upper limb skin metastases reappeared, the brain metastases relapsed, and she again experienced symptoms of meningitis carcinomatosa. Lapatinib was restarted, resulting in an immediate improvement in the symptoms and a reduction in the skin and brain metastases. Immunohistochemical staining of the lapatinib-sensitive metastatic skin tumor showed it to be HER2(2+), FISH(-)/HER1(-). This result suggested that the lapatinib-sensitive lesions in the brain and meninges were also HER2-positive. Carcinomatosa meningitis has a very poor prognosis and no effective treatment has yet been developed. Here, we report the first case in which lapatinib has been used to effectively treat meningitis carcinomatosa in HER2(-)/HER1(-) relapsed breast cancer.

Intratumoral CD8(+) T/FOXP3 (+) cell ratio is a predictive marker for survival in patients with colorectal cancer.

The human immune system consists of a balance between immune surveillance against non-self antigens and tolerance of self-antigens. CD8(+) T cells and CD4(+) regulatory T cells (Tregs) are the main players for immune surveillance and tolerance, respectively. We examined immunohistochemically the immunological balance at the tumor site using 94 surgically resected colorectal cancer tissues. Forkhead box P3 (FOXP3)(+) cells were considered to be Tregs in the present study. The number of intratumoral FOXP3(+) cells (itFOXP3(+) cells) was positively correlated with lymph node metastases (P = 0.030). itCD8(+) T/itFOXP3(+) cell ratio negatively correlated with pathological stages (P = 0.048). Next, relationship between the number of itCD8(+) T cells or itFOXP3(+) cells and survival prognosis in 94 patients who underwent a curative resection was analyzed. Only itCD8(+) T/itFOXP3(+) cell ratio positively correlated with disease-free survival (0.023) and overall survival (P = 0.010). Multivariate analysis indicated that itCD8(+) T/itFOXP3(+) cell ratio is an independent prognostic factor (P = 0.035) of overall survival. The number of itFOXP3(+) cells positively correlated with transforming growth factor-beta TGF-beta production at the tumor site (P = 0.020). In conclusion, itCD8(+) T/itFOXP3(+) cell ratio is a predictive marker for both disease-free survival time and overall survival time in patients with colorectal cancer. Importantly, itCD8(+) T/itFOXP3(+) cell ratio may be an independent prognostic factor. And, tumor-producing TGF-beta may contribute to the increased number of itFOXP3(+) cells.

Nuclear factor kappaB-activated monocytes contribute to pancreatic cancer progression through the production of Shh.

Recently, it was reported that Hh signaling is activated in tumor stromal cells but not in tumor cells themselves and that stromal cells may play a role in the proliferation of cancer cells. This suggests the possibility that stromal cells have an important role in the proliferation of tumor cells that may be mediated through Hh signaling. In this report, we present for the first time that inflammation-stimulated monocytes produce Shh through activation of the NF-kappaB signaling pathway, and that the Shh produced promotes the proliferation of pancreatic cancer cells in a paracrine manner through Hh signaling.

Gamma-secretase inhibitors enhance taxane-induced mitotic arrest and apoptosis in colon cancer cells.

BACKGROUND & AIMS: Colorectal cancers are resistant to conventional chemotherapeutic treatments, including taxanes. gamma-Secretase is a multimeric membrane protein complex responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. gamma-Secretase inhibitors have attracted increasing interest as anticancer drugs because of their ability to inhibit Notch signaling. However, the therapeutic usefulness of gamma-secretase inhibitors against colorectal cancers remains unclear. METHODS: The effects of gamma-secretase inhibitors on growth and apoptosis induced by various chemotherapeutic agents in colon cancer cells were evaluated using Hoechst 33342 staining, colony formation assay, and cell cycle analysis. The effect of gamma-secretase inhibitors on taxane-induced mitotic arrest was evaluated using the cyclin B1-associated histone H1 kinase assay and MPM-2 reactivity. The involvement of Notch signaling was evaluated by the silencing of Notch/CBF1 signaling by RNA interference. RESULTS: gamma-Secretase inhibitors enhanced taxane-induced mitotic arrest and apoptosis of colon cancer cells both in vitro and in vivo, although gamma-secretase inhibitors alone did not affect growth and apoptosis of colon cancer cells. We also showed that this effect by gamma-secretase inhibitors was restricted to taxanes and colon cancer cells. Silencing of Notch/CBF1 signaling failed to affect paclitaxel-induced mitotic arrest and apoptosis. CONCLUSIONS: These data suggest that gamma-secretase inhibitors could be a new therapeutic modality for overcoming resistance to taxanes in colorectal cancers.