Intra-tumoral heterogeneity in metastatic potential and survival signaling between iso-clonal HCT116 and HCT116b human colon carcinoma cell lines.

BACKGROUND: Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS). MATERIALS AND METHODS: HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days. RESULTS: Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death. CONCLUSIONS: We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer

Tumor Heterogeneity as a Predictor of Response to Neoadjuvant Chemotherapy in Locally Advanced Rectal Cancer

BACKGROUND: Neoadjuvant chemoradiotherapy (nCRT) is the standard of care for locally advanced adenocarcinoma of the rectum, but it is currently unknown which patients have disease that will respond. This study tested the correlation between response to nCRT and intratumoral heterogeneity using next-generation sequencing assays. PATIENTS AND METHODS: DNA was extracted from formalin-fixed, paraffin-embedded biopsy samples from a cohort of patients with locally advanced rectal adenocarcinoma (T3/4 or N1/2 disease) who received nCRT. High read-depth sequencing of \u3e 400 cancer-relevant genes was performed. Tumor mutations and variant allele frequencies were used to calculate mutant-allele tumor heterogeneity (MATH) scores as measures of intratumoral heterogeneity. Response to nCRT was pathologically scored after surgical resection. RESULTS: Biopsy samples from 21 patient tumors were analyzed. Eight patients had disease noted to have complete response, 2 moderate, 4 minimal, and 7 poor. Higher MATH scores correlated with poorer response to treatment, demonstrating significantly increased tumor heterogeneity compared to complete response (P = .039). CONCLUSION: The application of MATH scores as a measure of tumor heterogeneity may provide a useful biomarker for treatment response in locally advanced rectal cancer

Transforming growth factor-β suppresses metastasis in a subset of human colon carcinoma cells.

BACKGROUND: TGFβ signaling has typically been associated with suppression of tumor initiation while the role it plays in metastasis is generally associated with progression of malignancy. However, we present evidence here for an anti-metastatic role of TGFβ signaling. METHODS: To test the importance of TGFβ signaling to cell survival and metastasis we compared human colon carcinoma cell lines that are either non-tumorigenic with TGFβ response (FET), or tumorigenic with TGFβ response (FETα) or tumorigenic with abrogated TGFβ response via introduction of dominant negative TGFβRII (FETα/DN) and their ability to metastasize. Metastatic competency was assessed by orthotopic transplantation. Metastatic colony formation was assessed histologically and by imaging. RESULTS: Abrogation of TGFβ signaling through introduction of a dominant negative TGFβ receptor II (TGFβRII) in non-metastatic FETα human colon cancer cells permits metastasis to distal organs, but importantly does not reduce invasive behavior at the primary site. Loss of TGFβ signaling in FETα-DN cells generated enhanced cell survival capabilities in response to cellular stress in vitro. We show that enhanced cellular survival is associated with increased AKT phosphorylation and cytoplasmic expression of inhibitor of apoptosis (IAP) family members (survivin and XIAP) that elicit a cytoprotective effect through inhibition of caspases in response to stress. To confirm that TGFβ signaling is a metastasis suppressor, we rescued TGFβ signaling in CBS metastatic colon cancer cells that had lost TGFβ receptor expression due to epigenetic repression. Restoration of TGFβ signaling resulted in the inhibition of metastatic colony formation in distal organs by these cells. These results indicate that TGFβ signaling has an important role in the suppression of metastatic potential in tumors that have already progressed to the stage of an invasive carcinoma. CONCLUSIONS: The observations presented here indicate a metastasis suppressor role for TGFβ signaling in human colon cancer cells. This raises the concern that therapies targeting inhibition of TGFβ signaling may be imprudent in some patient populations with residual TGFβ tumor suppressor activity

Colorectal Cancer Incidence and Mortality Disparities in New Mexico

Background. Previous analyses indicated that New Mexican Hispanics and American Indians (AI) did not experience the declining colorectal cancer (CRC) incidence and mortality rates observed among non-Hispanic whites (NHW). We evaluated more recent data to determine whether racial/ethnic differences persisted. Methods. We used New Mexico Surveillance Epidemiology and End Results data from 1995 to 2009 to calculate age-specific incidence rates and age-adjusted incidence rates overall and by tumor stage. We calculated mortality rates using National Center for Health Statistics’ data. We used joinpoint regression to determine annual percentage change (APC) in age-adjusted incidence rates. Analyses were stratified by race/ethnicity and gender. Results. Incidence rates continued declining in NHW (APC −1.45% men, −1.06% women), while nonsignificantly increasing for AI (1.67% men, 1.26% women) and Hispanic women (0.24%). The APC initially increased in Hispanic men through 2001 (3.33%, P=0.06), before declining (−3.10%, P=0.003). Incidence rates declined in NHW and Hispanics aged 75 and older. Incidence rates for distant-stage cancer remained stable for all groups. Mortality rates declined significantly in NHW and Hispanics. Conclusions. Racial/ethnic disparities in CRC persist in New Mexico. Incidence differences could be related to risk factors or access to screening; mortality differences could be due to patterns of care for screening or treatment

Identification of a Novel TGFβ/PKA Signaling Transduceome in Mediating Control of Cell Survival and Metastasis in Colon Cancer

Understanding drivers for metastasis in human cancer is important for potential development of therapies to treat metastases. The role of loss of TGFβ tumor suppressor activities in the metastatic process is essentially unknown.Utilizing in vitro and in vivo techniques, we have shown that loss of TGFβ tumor suppressor signaling is necessary to allow the last step of the metastatic process - colonization of the metastatic site. This work demonstrates for the first time that TGFβ receptor reconstitution leads to decreased metastatic colonization. Moreover, we have identified a novel TGFβ/PKA tumor suppressor pathway that acts directly on a known cell survival mechanism that responds to stress with the survivin/XIAP dependent inhibition of caspases that effect apoptosis. The linkage between the TGFβ/PKA transduceome signaling and control of metastasis through induction of cell death was shown by TGFβ receptor restoration with reactivation of the TGFβ/PKA pathway in receptor deficient metastatic colon cancer cells leading to control of aberrant cell survival.This work impacts our understanding of the possible mechanisms that are critical to the growth and maintenance of metastases as well as understanding of a novel TGFβ function as a metastatic suppressor. These results raise the possibility that regeneration of attenuated TGFβ signaling would be an effective target in the treatment of metastasis. Our work indicates the clinical potential for developing anti-metastasis therapy based on inhibition of this very important aberrant cell survival mechanism by the multifaceted TGFβ/PKA transduceome induced pathway. Development of effective treatments for metastatic disease is a pressing need since metastases are the major cause of death in solid tumors

<i>γ</i>-ray Flux and Spectral Variability of Blazar Ton 599 during Its 2021 Flare

Blazars are known to emit exceptionally variable non-thermal emission over the wide range (from radio to γ-rays) of electromagnetic spectrum. We present here the results of our γ-ray flux and spectral variability study of the blazar Ton 599, which has been recently observed in the γ-ray flaring state. Using 0.1–300 GeV γ-ray data from the Fermi Gamma-ray Space Telescope (hereinafter Fermi), we generated one-day binned light curve of Ton 599 for a period of about one-year from MJD 59,093 to MJD 59,457. During this one year period, the maximum γ-ray flux detected was 2.24 ± 0.25 × 10−6 ph cm−2 s−1 at MJD 59,399.50. We identified three different flux states, namely, epoch A (quiescent), epoch B (pre-flare) and epoch C (main-flare). For each epoch, we calculated the γ-ray flux variability amplitude (Fvar) and found that the source showed largest flux variations in epoch C with Fvar∼ 35%. We modelled the γ-ray spectra for each epoch and found that the Log-parabola model adequately describes the γ-ray spectra for all the three epochs. We estimated the size of the γ-ray emitting region as 1.03 × 1016 cm and determined that the origin of γ-ray radiation, during the main-flare, could be outside of the broad line region

&gamma;-ray Flux and Spectral Variability of Blazar Ton 599 during Its 2021 Flare

Blazars are known to emit exceptionally variable non-thermal emission over the wide range (from radio to &gamma;-rays) of electromagnetic spectrum. We present here the results of our &gamma;-ray flux and spectral variability study of the blazar Ton 599, which has been recently observed in the &gamma;-ray flaring state. Using 0.1&ndash;300 GeV &gamma;-ray data from the Fermi Gamma-ray Space Telescope (hereinafter Fermi), we generated one-day binned light curve of Ton 599 for a period of about one-year from MJD 59,093 to MJD 59,457. During this one year period, the maximum &gamma;-ray flux detected was 2.24 &plusmn; 0.25 &times; 10&minus;6 ph cm&minus;2 s&minus;1 at MJD 59,399.50. We identified three different flux states, namely, epoch A (quiescent), epoch B (pre-flare) and epoch C (main-flare). For each epoch, we calculated the &gamma;-ray flux variability amplitude (Fvar) and found that the source showed largest flux variations in epoch C with Fvar&sim; 35%. We modelled the &gamma;-ray spectra for each epoch and found that the Log-parabola model adequately describes the &gamma;-ray spectra for all the three epochs. We estimated the size of the &gamma;-ray emitting region as 1.03 &times; 1016 cm and determined that the origin of &gamma;-ray radiation, during the main-flare, could be outside of the broad line region