Dysregulation of microRNA-34a expression causes drug-resistance to 5-FU in human colon cancer DLD-1 cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.

Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells.

Cancer-associated fibroblasts (CAFs), represent a pivotal compartment of solid cancers (desmoplasia), and are causatively implicated in cancer development and progression. CAFs are recruited by growth factors secreted by cancer cells and they present a myofibroblastic phenotype, similar to the one obtained by resident fibroblasts during wound healing. Paracrine signaling between cancer cells and CAFs results in a unique protein expression profile in areas of desmoplastic reaction, which is speculated to drive metastasis. In an attempt to decipher large-scale proteomic profiles of the cancer invasive margins, we developed an in vitro coculture model system, based on tumor-host cell interactions between colon cancer cells and CAFs. Proteomic analysis of conditioned media derived from these cocultures coupled to mass spectrometry and bioinformatic analysis was performed to uncover myofibroblastic signatures of the cancer invasion front. Our analysis resulted in the identification and generation of a desmoplastic protein dataset (DPD), consisting of 152 candidate proteins of desmoplasia. By using monoculture exclusion datasets, a secretome algorithm and gene-expression meta-analysis in DPD, we specified a 22-protein "myofibroblastic signature" with putative importance in the regulation of colorectal cancer metastasis. Of these proteins, we investigated collagen type XII by immunohistochemistry, a fibril-associated collagen with interrupted triple helices (FACIT), whose expression has not been reported in desmoplastic lesions in any type of cancer. Collagen type XII was highly expressed in desmoplastic stroma by and around alpha-smooth muscle actin (α-SMA) positive CAFs, as well as in cancer cells lining the invasion front, in a small cohort of colon cancer patients. Other stromal markers, such as collagen type III, were also expressed in stromal collagen, but not in cancer cells. In a complementary fashion, gene expression meta-analysis revealed that COL12A1 is also an upregulated gene in colorectal cancer. Our proteomic analysis identified previously documented markers of tumor invasion fronts and our DPD could serve as a pool for future investigation of the tumor microenvironment. Collagen type XII is a novel candidate marker of myofibroblasts, and/or cancer cells undergoing dedifferentiation.

/pdf/proteomic-signatures-of-the-desmoplastic-invasion-front-15tkhd4wxo.pdf

Proteomic signatures of the desmoplastic invasion front reveal collagen type XII as a marker of myofibroblastic differentiation during colorectal cancer metastasis.

Purpose: To evaluate the effects of single-nucleotide polymorphisms (SNP) in microRNA-related genes on clinical outcomes in patients with colorectal cancer (CRC) receiving first-line fluoropyrimidine-based chemotherapy. Experimental Design: Forty-one SNPs in 26 microRNA-related genes were genotyped in 1,097 patients with CRC recruited at the University of Texas MD Anderson Cancer Center (Houston, TX). Patients were enrolled between 1990 and 2008 and last follow-up was in 2010. The associations between genotypes and recurrence-free survival (RFS), progression-free survival (PFS), and overall survival (OS) stratified by clinical stage were analyzed in 741 newly diagnosed patients (diagnosed within 1 year) and replicated the findings in an additional 356 patients. Results: In patients with stage III disease, mir608 : rs4919510 was associated with increased risk for both recurrence [HR, 2.72; 95% confidence interval (CI), 1.38–5.33] and death (HR, 3.53; 95%CI, 1.42–8.73). The associations were confirmed in the replication set, and the combined HRs for training and replication sets were 1.65 (95% CI, 1.13–2.41) for recurrence and 1.96 (95% CI, 1.19–3.21) for death, respectively. The mir219 -1:rs213210 showed consistent association with death in the training set (HR, 3.86; 95% CI, 1.33–11.22), the replication set (HR, 3.33; 95% CI, 1.39–7.98), and combined data set (HR, 3.22; 95% CI, 1.70–6.10). In combined analysis of these two SNPs, patients carrying the variant genotypes at both sites exhibited a 5.6-fold increased risk of death. Conclusion: Genetic polymorphisms in the microRNA pathway may predict prognosis in patients with stage III CRC treated with fluoropyrimidine-based chemotherapy. Clin Cancer Res; 18(14); 3982–91. ©2012 AACR .

https://clincancerres.aacrjournals.org/content/clincanres/18/14/3982.full.pdf

Genetic Polymorphisms in MicroRNA-Related Genes as Predictors of Clinical Outcomes in Colorectal Adenocarcinoma Patients

Targeting p53 as a therapeutic strategy in sensitizing TRAIL-induced apoptosis in cancer cells.

Colorectal cancer is the third most frequent malignant neoplasm in Western countries. After complete resection, 5-year overall survival varies according to the initial stage. Adjuvant chemotherapy (CT) is indicated in patients with colon cancer at high-risk stage II, stage III and after complete resection of metastases. 5-Fluorouracil (5FU), alone or modulated with levamisol or leucovorin (LV), oral fluoropyrimidines, raltitrexed, irinotecan and oxaliplatin have been studied as adjuvant therapy for colon cancer. Nowadays, oxaliplatin-based regimens, FOLFOX or FLOX, are considered as the standard adjuvant CT. If there are contraindications for oxaliplatin, the best alternatives are capecitabine or continuous infusion of 5FU/LV. The role of monoclonal antibodies, cetuximab and bevacizumab, combined with oxaliplatin/fluoropyrimidine-based CT is under investigation in clinical trials. This article reviews the state of the art and the future perspectives of adjuvant therapy in colon cancer. Prognostic and predictive factors are also commented on.

Adjuvant chemotherapy for stages II, III and IV of colon cancer.

Hepatobiliary cystadenocarcinomas (BCACs) with mesenchymal stroma are a rare cystic lesion. This tumour needs to be distinguished from benign biliary cystadenoma, which is antecedent in most cases. The treatment of choice is radical excision of the mass. The diagnostic evaluation, surgical management, pathological characteristics, treatment and follow-up of one patient with hepatobiliary cystadenocarcinoma with ovarian stroma is described. Preoperative diagnosis of BCACs is often difficult, because their clinical manifestations are similar to those of other hepatic cystic lesions. MRI is suitable for accurate characterisation of cystic biliary lesions, but distinguishing between cystadenoma and cystadenocarcinoma remains difficult on the basis of imaging findings. Complete surgical excision gives a relatively good chance of long-term survival because of the slow growth rate of these tumours.

Biliary cystadenocarcinoma with mesenchymal stroma.

HER2-positive breast cancer (BC) is an aggressive subtype that affects 20–25% of BC patients. For these patients, neoadjuvant therapy is a good option that targets a pathological complete response (pCR) and more breast-conserving surgery. In effect, the outcomes of patients with HER2-positive BC have dramatically improved since the introduction of anti-HER2 antibodies such as trastuzumab (TZ) and/or pertuzumab (PZ) added to chemotherapy. This study sought to examine whether correlation exists between copy number variations (CNVs) in several genes related to the PI3K/AKT pathway (HER2, FGFR1, PIK3CA, AKT3 and MDM2) and the efficacy of anti-HER2 neoadjuvant treatment in patients with early HER2-positive BC. Forty-nine patients received TZ or PZ/TZ and chemotherapy as neoadjuvant treatment. Gene CNVs were determined by quantitative polymerase chain reaction on paraffin-embedded biopsy specimens. The response to 6 months of therapy was assessed by Miller–Payne grading of the tumor on surgical resection; grades 4 and 5, indicating >90% tumor reduction, were defined as a good response. A good response was shown by 64.5% and a pCR by 31.2% of patients. When stratified by anti-HER2 antibody received and gene CNV, it was found that patients with FGFR1 gene amplification or those with FGFR1 amplification treated with TZ alone showed a poor response (p = 0.024 and p = 0.037, respectively). In the subset of patients treated with TZ/PZ combined, the pCR rate was significantly lower among those showing FGFR1 amplification (p = 0.021). Although based on a small sample size, our findings suggest that patients with FGFR1 amplification might benefit less from anti-HER2 antibody therapy.

/pdf/fgfr1-amplification-and-response-to-neoadjuvant-anti-her2-syc2fzg5.pdf

FGFR1 Amplification and Response to Neoadjuvant Anti-HER2 Treatment in Early HER2-Positive Breast Cancer


 Background: Novel therapies are needed upon progression to first-line CDK4/6 inhibitor (CDKi) plus endocrine therapy in advanced HR+ breast cancer. One frequent alteration driving resistance to CDKi plus hormones is FGFR1/2 amplification/overexpression. We have demonstrated that a combined assay of RNAScope and FISH, detecting RNA overexpression and genomic amplification of FGFR1/2, captures more FGFR-aberrant cases than either test alone. When these alterations exist, the triple estrogen receptor (ER), CDK4/6 and FGFR1/2 blockade is able to revert acquired resistance to CDKi (Breast Cancer Res; 23:21-37; 2021). Rogaratinib is a selective FGFR1-4 kinase inhibitor with significant activity in FGFR-aberrant malignancies. In this trial we will test the safety of the triple blockade combining fulvestrant, palbociclib and rogaratinib. We will also study its role as a resistant-reversion combo for FGFR1/2-positive, HR+ patients progressing to a CDK4/6 inhibitor plus aromatase inhibitor (AI) in the first-line metastatic setting. Trial design: Single-arm, prospective, multicentric, open-label, phase I dose-escalation trial. Patients will be pre-screened for amplification and/or overexpression of FGFR1/2 by FISH and RNAScope, during their first-line treatment. Positive patients will start treatment: on day 1, patients will receive rogaratinib q12 hours. On day 2, patients will continue on rogaratinib, receive fulvestrant (500 ug IM) and start on palbociclib (100 mg/day on days 1-21). A second fulvestrant dose (500 ug) will be administered in day +15. The first cycle will be 29 days, followed by 28-day cycles (continuous rogaratinib q12 hours days 1-28, fulvestrant 500 ug day 1, and palbociclib 100 mg/day days 1-21). Rogaratinib will be escalated, starting on 400 mg/bid in 200 mg/bid increments, following a classic 3+3 schedule. Patients experiencing ≤ grade 1 tolerable side effects as the greatest toxicity will be allowed to escalate palbociclib to 125 in cycle 2. A full pharmacokinetic profile and pharmacodynamics (plasma FGF23 and phosphate levels) will be performed on days 1 and 15 of cycle 1. The impact of the triple combination in immunodynamics will be assessed on PBMCs on days 2 and 15. Ten additional HR+, FGFR1/2 + patients will be accrued at the RP2D in an expansion cohort to study efficacy. RECIST 1.1 and NCI CTC AE V 5.0 criteria will be used for assessing disease control (q8 weeks) and toxicity. Eligibility criteria: Pre-screening: Women >18 year old diagnosed with advanced, non-curable HR+ breast cancer; 2) who are receiving first-line treatment with an AI and any oral CDKi; 3) Adequate organ function. Full screening: 4) Positivity for amplification (FISH ratio>2.2) and/or overexpression (RNAScope score of 3+ or 4+) of either FGFR1/2. 5) PD to first-line AI + CDKi within the last 28 days. 6) Recovery from previous toxicity to < tolerable G2. Specific aims: Primary: 1) To determine RP2D of the triple combination of fulvestrant, palbociclib and rogaratinib. 2) To assess the safety and toxicity of the former combination 3) To study the pharmacokinetics, pharmacodynamics and immunodynamics of the triplet. Secondary: 1) To determine the PFS time and 6-month PFS rate of the triplet in FGFR1/2 amplified/overexpressed patients AI + CDKi doublet. Statistical methods: We will follow a 3+3 escalation schedule; thus, the minimum and maximum number of patients for the dose-escalation phase will range between 6 and 18 (no escalation planned beyond 800 mg/bid rogaratinib). Ten additional patients will be accrued in the expansion cohort (N=28). The anticipated rate of positive screening is 40%; thus, we will screen approximately 70 patients. . Present accrual/target accrual: 55/70 screened; 16 FGFR1/2-positive (of 28 planned); 2 accrued and treated.
 Citation Format: Sonia Pernas, Cristina Hernando, Begoña Bermejo, Noelia Martinez-Jañez, Jose A García Saenz, Serafin Morales, Luis Manso, Jorge Silva, Juan A Guerra, Diego Malon, Silvana Mouron, Eduardo Caleiras, Miguel Quintela-Fandino. Rogaratinib, palbociclib and fulvestrant in advanced hormone receptor-positive (HR+), FGFR1/2-positive breast cancer: Phase I trial plus an expansion cohort [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-20-01.

Abstract OT2-20-01: Rogaratinib, palbociclib and fulvestrant in advanced hormone receptor-positive (HR+), FGFR1/2-positive breast cancer: Phase I trial plus an expansion cohort

Abstract CDK4/6 inhibitors benefit a minority of patients who receive them in the breast cancer adjuvant setting. p27Kip1 is a protein that inhibits CDK/Cyclin complexes. We hypothesized that single-nucleotide polymorphisms that impaired p27Kip1 function could render patients refractory to endocrine therapy but responsive to CDK4/6 inhibitors, narrowing the patient subpopulation that requires CDK4/6 inhibitors. We found that the p27Kip1 V109G single-nucleotide polymorphism is homozygous in approximately 15% of hormone-positive breast cancer patients. Polymorphic patients experience rapid failure in response to endocrine monotherapy compared with wild-type or heterozygous patients in the first-line metastatic setting (progression-free survival: 92 vs 485 days, P < .001); when CDK4/6 inhibitors are added, the differences disappear (progression-free survival: 658 vs 761 days, P = .92). As opposed to wild-type p27Kip1, p27Kip1 V109G is unable to suppress the kinase activity of CDK4 in the presence of endocrine inhibitors; however, palbociclib blocks CDK4 kinase activity regardless of the p27Kip1 status. p27Kip1 genotyping could constitute a tool for treatment selection.

/pdf/p27kip1-v109g-as-a-biomarker-for-cdk4-6-inhibitors-dhfj4oub.pdf

Diego Malón

Papers

Adjuvant chemotherapy for stages II, III and IV of colon cancer.

Biliary cystadenocarcinoma with mesenchymal stroma.

FGFR1 Amplification and Response to Neoadjuvant Anti-HER2 Treatment in Early HER2-Positive Breast Cancer

Abstract OT2-20-01: Rogaratinib, palbociclib and fulvestrant in advanced hormone receptor-positive (HR+), FGFR1/2-positive breast cancer: Phase I trial plus an expansion cohort

p27Kip1 V109G as a biomarker for CDK4/6 inhibitors indication in hormone receptor–positive breast cancer