Masayoshi Iwamoto

The role of CXCR3 and CXCR4 in colorectal cancer metastasis

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis. We previously demonstrated that forced expression of CXCR3 promotes colon cancer metastasis preferentially to the draining lymph nodes (LNs), with poor prognosis. Using clinical colorectal cancer (CRC) samples, here, we show that expressions of CXCR3 and CXCR4 are significantly higher in metastatic foci within LNs and liver compared to primary tumors, whereas ligands for CXCR3 and CXCR4 are not. We also have demonstrated that some human CRC cell lines constitutively express both CXCR3 and CXCR4, and that activation of CXCR3 strengthens the CXCR4‐mediated cell migration in vitro in a synergistic manner. By constructing SW620 cell lines with reduced expression of CXCR3 and/or CXCR4 using microRNA, we investigated in vivo metastatic activities in a mouse rectal transplantation model. Six weeks after inoculation, CXCR3‐, CXCR4‐, and CXCR3/CXCR4 double‐knockdowns significantly reduced metastasis to LNs, liver and lungs, compared to the control (p < 0.05). Importantly, its suppressive effect on LN metastasis was significantly stronger in CXCR3‐ and CXCR3/CXCR4 double‐knockdowns. In addition, CXCR3‐ and CXCR3/CXCR4 double‐knockdowns significantly decreased the dissemination of cancer cells to liver and lungs, even after 2 weeks. These results indicate that targeting CXCR3 and CXCR4 can be a promising therapy against CRC metastasis.

Regulation of 18F-FDG Accumulation in Colorectal Cancer Cells with Mutated KRAS

KRAS gene mutations occur in approximately 40% of colorectal cancers (CRCs) and are associated with resistance to anti–epidermal growth factor receptor antibody therapy. We previously demonstrated that 18F-FDG accumulation in PET was significantly higher in CRCs with mutated KRAS than in those with wild-type KRAS in a clinical setting. Here, we investigated the mechanisms by which mutated KRAS increased 18F-FDG accumulation. Methods: Using paired isogenic human CRC cell lines that differ only in the mutational status of the KRAS gene, we measured 18F-FDG accumulation in these cells in vitro and in vivo. We also investigated the roles of proteins that have a function in 18F-FDG accumulation. Finally, we examined the relationship among mutated KRAS, hypoxia-inducible factor 1α (HIF-1α), and maximum standardized uptake value with 51 clinical CRC samples. Results: In the in vitro experiments, 18F-FDG accumulation was significantly higher in KRAS-mutant cells than in wild-type controls under normoxic conditions. The expression levels of glucose transporter 1 (GLUT1) and hexokinase type 2 (HK2) were higher in KRAS-mutant cells, and 18F-FDG accumulation was decreased by knockdown of GLUT1. Hypoxic induction of HIF-1α was higher in KRAS-mutant cells than in wild-type controls; in turn, elevated HIF-1α resulted in higher GLUT1 expression and 18F-FDG accumulation. In addition, HIF-1α knockdown decreased 18F-FDG accumulation under hypoxic conditions only in the KRAS-mutant cells. Small-animal PET scans showed in vivo 18F-FDG accumulation to be significantly higher in xenografts with mutated KRAS than in those with wild-type KRAS. The immunohistochemistry of these xenograft tumors showed that staining of GLUT1 was consistent with that of HIF-1α and pimonidazole. In a retrospective analysis of clinical samples, KRAS mutation exhibited a significantly positive correlation with expressions of GLUT1 and HIF-1α and with maximum standardized uptake value. Conclusion: Mutated KRAS caused higher 18F-FDG accumulation possibly by upregulation of GLUT1; moreover, HIF-1α additively increased 18F-FDG accumulation in hypoxic lesions. 18F-FDG PET might be useful for predicting the KRAS status noninvasively.

Loss of SMAD4 Promotes Colorectal Cancer Progression by Accumulation of Myeloid-Derived Suppressor Cells through the CCL15–CCR1 Chemokine Axis

Purpose: We previously reported that loss of SMAD4 promotes chemokine CCL15 expression to recruit CCR1+ myeloid cells via the CCL15–CCR1 axis, which facilitates metastasis of colorectal cancer to the liver. The purposes of this study were to investigate whether essentially the same mechanism works in tumor invasion of the primary colorectal cancer and to evaluate the clinical importance of CCL15 expression and CCR1+ cell accumulation. Experimental Design: Using human colorectal cancer cell lines with reduced expression of SMAD4 or CCL15, we investigated tumor growth activities in vivo. We used immunohistochemistry (IHC) to investigate expression of SMAD4, CCL15, and CCR1 with 333 clinical specimens of primary colorectal cancer. We next characterized the CCR1+ cells using double immunofluorescence staining with several specific cell-type markers. Finally, we determined the serum CCL15 levels in 132 colorectal cancer patients. Results: In an orthotopic xenograft model, CCL15 secreted from SMAD4-deficient colorectal cancer cells recruited CCR1+ cells, resulting in aggressive tumor growth. IHC indicated that loss of SMAD4 was significantly associated with CCL15 expression, and that CCL15-positive primary colorectal cancers recruited approximately 2.2 times more numbers of CCR1+ cells at their invasion front than CCL15-negative colorectal cancers. Importantly, these CCR1+ cells were of the myeloid-derived suppressor cell (MDSC) phenotype (CD11b+, CD33+, and HLA-DR−). Most CCR1+ cells showed the granulocytic-MDSC phenotype (CD15+), whereas some showed the monocytic-MDSC phenotype (CD14+). Serum CCL15 levels in colorectal cancer patients were significantly higher than in controls. Conclusions: Blocking the recruitment of CCR1+ MDSCs may represent a novel molecular-targeted therapy, and serum CCL15 concentration can be a novel biomarker for colorectal cancer. Clin Cancer Res; 22(2); 492–501. ©2015 AACR.

Molecular mechanisms of liver metastasis

Colorectal cancer is the second most common cancer, and is the third leading cause of cancer-related death in Japan. The majority of these deaths is attributable to liver metastasis. Recent studies have provided increasing evidence that the chemokine–chemokine receptor system is a potential mechanism of tumor metastasis via multiple complementary actions: (a) by promoting cancer cell migration, invasion, survival and angiogenesis; and (b) by recruiting distal stromal cells (i.e., myeloid bone marrow-derived cells) to indirectly facilitate tumor invasion and metastasis. Here, we discuss recent preclinical and clinical data supporting the view that chemokine pathways are potential therapeutic targets for liver metastasis of colorectal cancer.

Relationship Between 18F-FDG PET/CT Scans and KRAS Mutations in Metastatic Colorectal Cancer

Several studies have shown that KRAS mutations in colorectal cancer (CRC) result in the lack of response to anti-epidermal growth factor receptor–based therapy; thus, KRAS mutational testing has been incorporated into routine clinical practice. However, 1 limitation of this test is the heterogeneity of KRAS status, which can be either intratumoral heterogeneity within an individual primary CRC or discordant KRAS status between a primary CRC and its corresponding metastases. We previously reported that 18F-FDG accumulation was significantly higher in primary CRCs with mutated KRAS than in those with wild-type KRAS. However, the clinical utility of the previous report has been limited because endoscopic biopsy for testing KRAS status is safe and feasible only in primary CRC. The purpose of this study was to investigate whether KRAS status is associated with 18F-FDG accumulation in metastatic CRC and whether 18F-FDG PET/CT scans can be used to predict the KRAS status of metastatic CRC. Methods: A retrospective analysis was performed on 55 metastatic CRC tumors that were identified by 18F-FDG PET/CT before surgical resection. Maximum standardized uptake value (SUVmax) of the respective metastatic tumor was calculated from 18F-FDG accumulation. Results: From the analysis with the 55 tumors, no significant correlation was found between SUVmax and KRAS status. We next analyzed only tumors larger than 10 mm to minimize the bias of partial-volume effect and found that SUVmax was significantly higher in the KRAS-mutated group than in the wild-type group (8.3 ± 4.1 vs. 5.7 ± 2.4, respectively; P = 0.03). Multivariate analysis indicated that SUVmax remained significantly associated with KRAS mutations (P = 0.04). KRAS status could be predicted with an accuracy of 71.4% when an SUVmax cutoff value of 6.0 was used. Conclusion: 18F-FDG accumulation into metastatic CRC was associated with KRAS status. 18F-FDG PET/CT scans may be useful for predicting the KRAS status of metastatic CRC and help in determining the therapeutic strategies against metastatic CRC.

Metabolic Alterations Caused by KRAS Mutations in Colorectal Cancer Contribute to Cell Adaptation to Glutamine Depletion by Upregulation of Asparagine Synthetase

A number of clinical trials have shown that KRAS mutations of colorectal cancer (CRC) can predict a lack of responses to anti-epidermal growth factor receptor–based therapy. Recently, there have been several studies to elucidate metabolism reprogramming in cancer. However, it remains to be investigated how mutated KRAS can coordinate the metabolic shift to sustain CRC tumor growth. In this study, we found that KRAS mutation in CRC caused alteration in amino acid metabolism. KRAS mutation causes a marked decrease in aspartate level and an increase in asparagine level in CRC. Using several human CRC cell lines and clinical specimens of primary CRC, we demonstrated that the expression of asparagine synthetase (ASNS), an enzyme that synthesizes asparagine from aspartate, was upregulated by mutated KRAS and that ASNS expression was induced by KRAS-activated signaling pathway, in particular PI3K-AKT-mTOR pathway. Importantly, we demonstrated that KRAS-mutant CRC cells could become adaptive to glutamine depletion through asparagine biosynthesis by ASNS and that asparagine addition could rescue the inhibited growth and viability of cells grown under the glutamine-free condition in vitro. Notably, a pronounced growth suppression of KRAS-mutant CRC was observed upon ASNS knockdown in vivo. Furthermore, combination of L-asparaginase plus rapamycin markedly suppressed the growth of KRAS-mutant CRC xenografts in vivo, whereas either L-asparaginase or rapamycin alone was not effective. These results indicate ASNS might be a novel therapeutic target against CRCs with mutated KRAS.

Clinical Role of ASCT2 (SLC1A5) in KRAS-Mutated Colorectal Cancer

Mutation in the KRAS gene induces prominent metabolic changes. We have recently reported that KRAS mutations in colorectal cancer (CRC) cause alterations in amino acid metabolism. However, it remains to be investigated which amino acid transporter can be regulated by mutated KRAS in CRC. Here, we performed a screening of amino acid transporters using quantitative reverse-transcription polymerase chain reaction (RT-PCR) and then identified that ASCT2 (SLC1A5) was up-regulated through KRAS signaling. Next, immunohistochemical analysis of 93 primary CRC specimens revealed that there was a significant correlation between KRAS mutational status and ASCT2 expression. In addition, the expression level of ASCT2 was significantly associated with tumor depth and vascular invasion in KRAS-mutant CRC. Notably, significant growth suppression and elevated apoptosis were observed in KRAS-mutant CRC cells upon SLC1A5-knockdown. ASCT2 is generally known to be a glutamine transporter. Interestingly, SLC1A5-knockdown exhibited a more suppressive effect on cell growth than glutamine depletion. Furthermore, SLC1A5-knockdown also resulted in the suppression of cell migration. These results indicated that ASCT2 (SLC1A5) could be a novel therapeutic target against KRAS-mutant CRC.

Loss of SMAD4 Promotes Lung Metastasis of Colorectal Cancer by Accumulation of CCR1+ Tumor-Associated Neutrophils through CCL15-CCR1 Axis

Purpose: We have reported loss of SMAD4 promotes expression of CCL15 from colorectal cancer to recruit CCR1+ myeloid cells through the CCL15-CCR1 axis, which contributes to invasion and liver metastasis. However, the molecular mechanism of lung metastasis is yet to be elucidated. Our purpose is to determine whether similar mechanism is involved in the lung metastasis of colorectal cancer. Experimental Design: In a mouse model, we examined whether SMAD4 could affect the metastatic activity of colorectal cancer cells to the lung through the CCL15-CCR1 axis. We immunohistochemically analyzed expression of SMAD4, CCL15, and CCR1 with 107 clinical specimens of colorectal cancer lung metastases. We also characterized the CCR1+ myeloid cells using several cell-type–specific markers. Results: In a mouse model, CCL15 secreted from SMAD4-deficient colorectal cancer cells recruited CCR1+ cells, promoting their metastatic activities to the lung. Immunohistochemical analysis of lung metastases from colorectal cancer patients revealed that CCL15 expression was significantly correlated with loss of SMAD4, and that CCL15-positive metastases recruited approximately 1.9 times more numbers of CCR1+ cells than CCL15-negative metastases. Importantly, patients with CCL15-positive metastases showed a significantly shorter relapse-free survival (RFS) than those with CCL15-negative metastases, and multivariate analysis indicated that CCL15 expression was an independent predictor of shorter RFS. Immunofluorescent staining showed that most CCR1+ cells around lung metastases were tumor-associated neutrophil, although a minor fraction was granulocytic myeloid-derived suppressor cell. Conclusions: CCL15-CCR1 axis may be a therapeutic target to prevent colorectal cancer lung metastasis. CCL15 can be a biomarker indicating poor prognosis of colorectal cancer patients with lung metastases. Clin Cancer Res; 23(3); 833–44. ©2016 AACR.

Mechanisms underlying 18F-fluorodeoxyglucose accumulation in colorectal cancer

Positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) is a diagnostic tool to evaluate metabolic activity by measuring accumulation of FDG, an analogue of glucose, and has been widely used for detecting small tumors, monitoring treatment response and predicting patients’ prognosis in a variety of cancers. However, the molecular mechanism of FDG accumulation into tumors remains to be investigated. It is well-known that most cancers are metabolically active with elevated glucose metabolism, a phenomenon known as the Warburg effect. The underlying mechanisms for elevated glucose metabolism in cancer tissues are complex. Recent reports have indicated the potential of FDG-PET/CT scans in predicting mutational status (e.g., KRAS gene mutation) of colorectal cancer (CRC), which suggests that FDG-PET/CT scans may play a key role in determining therapeutic strategies by non-invasively predicting treatment response to anti-epidermal growth factor receptor (EGFR) therapy. In this review, we summarize the current findings investigating the molecular mechanism of 18F-FDG accumulation in CRC.

Adenocarcinoma arising at a colostomy site with inguinal lymph node metastasis: report of a case

Inguinal lymph node metastasis from adenocarcinoma arising at a colostomy site is extremely rare, and the significance of surgical resection for metastatic inguinal lymph nodes has not been established. An 82-year-old woman who had undergone abdominoperineal resection 27 years earlier was admitted to our hospital complaining of bleeding from a colostomy. Physical examination revealed that a tumor at the colostomy site directly invaded into the peristomal skin, and that a left inguinal lymph node was firm and swollen. Positron emission tomography/computed tomography scan demonstrated accumulation of (18)F-fluorodeoxy glucose into both the colostomy tumor and the left swollen inguinal lymph node, while there was no evidence of metastasis to liver or lungs. She underwent open left hemicolectomy with wide local resection of the colostomy, and dissection of left inguinal lymph nodes. Histological diagnosis was a moderately differentiated adenocarcinoma that directly invaded into the surrounding skin and metastasized to the left inguinal lymph node. The patient has been followed up for >5 years without any sign of recurrence. In general, inguinal lymph node metastasis from colorectal cancers is regarded as a systemic disease with a poor prognosis, and so systemic chemotherapy and radiotherapy, but not surgical lymph node dissection, are recommended. Considering the lymphatic drainage route in the present case, inguinal lymph node metastasis does not represent a systemic disease but rather a sentinel nodal metastasis from adenocarcinoma at a colostomy site. Surgical dissection of metastatic inguinal lymph nodes should be considered to enable a favorable prognosis in the absence of distant metastasis to other organs.