Phosphorylation of the LCB1 subunit of Arabidopsis serine palmitoyltransferase stimulates its activity and modulates sphingolipid biosynthesis.

Sphingolipids are the structural components of membrane lipid bilayers and act as signaling molecules in many cellular processes. Serine palmitoyltransferase (SPT) is the first committed and rate-limiting enzyme in the de novo sphingolipids biosynthetic pathway. The core SPT enzyme is a heterodimer consisting of LONG-CHAIN BASE1 (LCB1) and LCB2 subunits. SPT activity is inhibited by orosomucoid proteins and stimulated by small subunits of SPT (ssSPTs). However, whether LCB1 is modified and how such modification might regulate SPT activity have to date been unclear. Here, we show that activation of MITOGEN-ACTIVATED PROTEIN KINASE 3 (MPK3) and MPK6 by upstream MKK9 and treatment with Flg22 (a pathogen-associated molecular pattern) increases SPT activity and induces the accumulation of sphingosine long-chain base t18:0 in Arabidopsis thaliana, with activated MPK3 and MPK6 phosphorylating AtLCB1. Phosphorylation of AtLCB1 strengthened its binding with AtLCB2b, promoted its binding with ssSPTs, and stimulated the formation of higher order oligomeric and active SPT complexes. Our findings therefore suggest a novel regulatory mechanism for SPT activity.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

Despite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition. IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as "O antigen." This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.

Gap junctions mediate glucose transfer to promote colon cancer growth in three-dimensional spheroid culture.

In vivo tumor growth is characterized by a necrotic core generated by oxygen and nutrients gradients, which is replicated by in vitro three-dimensional (3D) tumor spheroids but not traditional two-dimensional cell monolayers. Gap junctions provide direct communication between adjacent cells and play a critical role in cancer development, but their effects are still debatable. In this study, we found that connexin 43 (Cx43) reduced the area of necrotic core in colon cancer 3D spheroids, thus providing a growth advantage. This impact is dependent on gap junction channel functions, as the channel blocker carbenoxolone or connexin channel death mutant reverses this effect. Additionally, enhanced glucose uptake was detected in Cx43-overexpressing spheroids, along with upregulated mTOR, downregulated AMPK signaling, increased ATP content, and enhanced oxygen consumption rate. Furthermore, the xenograft mouse model confirmed the growth advantage of Cx43 in vivo. RNAseq data and clinical information from The Cancer Genome Atlas (TCGA) database indicated a more heterogeneous expression pattern of Cx43 in colon cancer compared to normal colon tissue, and higher Cx43 level is associated with worse clinical outcomes. Our data suggest a novel function of connexin in tumor growth, that gap junctions may provide nutrients transmitting routes in lieu of vasculature to meet the increasing metabolic requirement of solid tumors.

The HNF4α-BC200-FMR1-Positive Feedback Loop Promotes Growth and Metastasis in Invasive Mucinous Lung Adenocarcinoma.

Invasive mucinous lung adenocarcinoma (IMA) is a subtype of lung adenocarcinoma with a strong invasive ability. IMA frequently carries "undruggable" KRAS mutations, highlighting the need for new molecular targets and therapies. Nuclear receptor HNF4α is abnormally enriched in IMA, but the potential of HNF4α to be a therapeutic target for IMA remains unknown. Here, we report that P2 promoter-driven HNF4α expression promotes IMA growth and metastasis. Mechanistically, HNF4α transactivated lncRNA BC200, which acted as a scaffold for mRNA binding protein FMR1. BC200 promoted the ability of FMR1 to bind and regulate stability of cancer-related mRNAs and HNF4α mRNA, forming a positive feedback circuit. Mycophenolic acid, the active metabolite of FDA-approved drug mycophenolate mofetil, was identified as an HNF4α antagonist exhibiting anti-IMA activities in vitro and in vivo. This study reveals the role of a HNF4α-BC200-FMR1-positive feedback loop in promoting mRNA stability during IMA progression and metastasis, providing a targeted therapeutic strategy for IMA. SIGNIFICANCE: Growth and metastatic progression of invasive mucinous lung adenocarcinoma can be restricted by targeting HNF4α, a critical regulator of a BC200-FMR1-mRNA stability axis.

Deregulation of Exo70 Facilitates Innate and Acquired Cisplatin Resistance in Epithelial Ovarian Cancer by Promoting Cisplatin Efflux.

Whilst researches elucidating a diversity of intracellular mechanisms, platinum-resistant epithelial ovarian cancer (EOC) remains a major challenge in the treatment of ovarian cancer. Here we report that Exo70, a key subunit of the exocyst complex, contributes to both innate and acquired cisplatin resistance of EOC. Upregulation of Exo70 is observed in EOC tissues and is related to platinum resistance and progression-free survival of EOC patients. Exo70 suppressed the cisplatin sensitivity of EOC cells through promoting exocytosis-mediated efflux of cisplatin. Moreover, cisplatin-induced autophagy-lysosomal degradation of Exo70 protein by modulating phosphorylation of AMPK and mTOR, thereby reducing the cellular resistance. However, the function was hampered during prolonged cisplatin treatment, which in turn stabilized Exo70 to facilitate the acquired cisplatin resistance of EOC cells. Knockdown of Exo70, or inhibiting exocytosis by Exo70 inhibitor Endosidin2, reversed the cisplatin resistance of EOC cells both in vitro and in vivo. Our results suggest that Exo70 overexpression and excessive stability contribute to innate and acquired cisplatin resistance through the increase in cisplatin efflux, and targeting Exo70 might be an approach to overcome cisplatin resistance in EOC treatment.

Bacterial Redox Potential Powers Controlled Radical Polymerization.

Microbes employ a remarkably intricate electron transport system to extract energy from the environment. The respiratory cascade of bacteria culminates in the terminal transfer of electrons onto higher redox potential acceptors in the extracellular space. This general and inducible mechanism of electron efflux during normal bacterial proliferation leads to a characteristic fall in bulk redox potential (Eh), the degree of which is dependent on growth phase, the microbial taxa, and their physiology. Here, we show that the general reducing power of bacteria can be subverted to induce the abiotic production of a carbon-centered radical species for targeted bioorthogonal molecular synthesis. Using two species, Escherichia coli and Salmonella enterica serovar Typhimurium as model microbes, a common redox active aryldiazonium salt is employed to intervene in the terminal respiratory electron flow, affording radical production that is mediated by native redox-active molecular shuttles and active bacterial metabolism. The aryl radicals are harnessed to initiate and sustain a bioorthogonal controlled radical polymerization via reversible addition-fragmentation chain transfer (BacRAFT), yielding a synthetic extracellular matrix of "living" vinyl polymers with predetermined molecular weight and low dispersity. The ability to interface the ubiquitous reducing power of bacteria into synthetic materials design offers a new means for creating engineered living materials with promising adaptive and self-regenerative capabilities.

Structure-Activity Relationship Study Enables the Discovery of a Novel Berberine Analogue as the RXRα Activator to Inhibit Colon Cancer.

We reported recently that berberine (Ber), a traditional oriental medicine to treat gastroenteritis, binds and activates retinoid X receptor α (RXRα) for suppressing the growth of colon cancer cells. Here, we extended our studies based on the binding mode of Ber with RXRα by design, synthesis, and biological evaluation of a focused library of 15 novel Ber analogues. Among them, 3,9-dimethoxy-5,6-dihydroisoquinolino[3,2-a]isoquinolin-7-ium chloride (B-12) was identified as the optimal RXRα activator. More efficiently than Ber, B-12 bound and altered the conformation of RXRα/LBD, thereby suppressing the Wnt/ß-catenin pathway and colon cancer cell growth via RXRα mediation. In addition, B-12 not only preserved Ber's tumor selectivity but also greatly improved its bioavailability. Remarkably, in mice, B-12 did not show obvious side effects including hypertriglyceridemia as other RXRα agonists or induce hepatorenal toxicity. Together, our study describes an approach for the rational design of Ber-derived RXRα activators as novel effective antineoplastic agents for colon cancer.

ULK1 phosphorylates Exo70 to suppress breast cancer metastasis.

Increased expression of protein kinase ULK1 was reported to negatively correlate with breast cancer metastasis. Here we report that ULK1 suppresses the migration and invasion of human breast cancer cells. The suppressive effect is mediated through direct phosphorylation of Exo70, a key component of the exocyst complex. ULK1 phosphorylation inhibits Exo70 homo-oligomerization as well as its assembly to the exocyst complex, which are needed for cell protrusion formation and matrix metalloproteinases secretion during cell invasion. Reversely, upon growth factor stimulation, Exo70 is phosphorylated by ERK1/2, which in turn suppresses its phosphorylation by ULK1. Together, our study identifies Exo70 as a substrate of ULK1 that inhibits cancer metastasis, and demonstrates that two counteractive regulatory mechanisms are well orchestrated during tumor cell invasion.

Matrine Reverses the Warburg Effect and Suppresses Colon Cancer Cell Growth via Negatively Regulating HIF-1α.

The Warburg effect is a peculiar feature of cancer's metabolism, which is an attractive therapeutic target that could aim tumor cells while sparing normal tissue. Matrine is an alkaloid extracted from the herb root of a traditional Chinese medicine, Sophora flavescens Ait. Matrine has been reported to have selective cytotoxicity toward cancer cells but with elusive mechanisms. Here, we reported that matrine was able to reverse the Warburg effect (inhibiting glucose uptake and lactate production) and suppress the growth of human colon cancer cells in vitro and in vivo. Mechanistically, we revealed that matrine significantly decreased the messenger RNA (mRNA) and protein expression of HIF-1α, a critical transcription factor in reprogramming cancer metabolism toward the Warburg effect. As a result, the expression levels of GLUT1, HK2, and LDHA, the downstream targets of HIF-1α in regulating glucose metabolism, were dramatically inhibited by matrine. Moreover, this inhibitory effect of matrine was significantly attenuated when HIF-1α was knocked down or exogenous overexpressed in colon cancer cells. Together, our results revealed that matrine inhibits colon cancer cell growth via suppression of HIF-1α expression and its downstream regulation of Warburg effect. Matrine could be further developed as an antitumor agent targeting the HIF-1α-mediated Warburg effect for colon cancer treatment.

IL-23 costimulates antigen-specific MAIT cell activation and enables vaccination against bacterial infection.

Mucosal-associated invariant T (MAIT) cells are activated in a TCR-dependent manner by antigens derived from the riboflavin synthesis pathway, including 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), bound to MHC-related protein-1 (MR1). However, MAIT cell activation in vivo has not been studied in detail. Here, we have found and characterized additional molecular signals required for optimal activation and expansion of MAIT cells after pulmonary Legionella or Salmonella infection in mice. We show that either bone marrow-derived APCs or non-bone marrow-derived cells can activate MAIT cells in vivo, depending on the pathogen. Optimal MAIT cell activation in vivo requires signaling through the inducible T cell costimulator (ICOS), which is highly expressed on MAIT cells. Subsequent expansion and maintenance of MAIT-17/1-type responses are dependent on IL-23. Vaccination with IL-23 plus 5-OP-RU augments MAIT cell-mediated control of pulmonary Legionella infection. These findings reveal cellular and molecular targets for manipulating MAIT cell function under physiological conditions.

Comparative phosphoproteomic analysis of developing maize seeds suggests a pivotal role for enolase in promoting starch synthesis.

Maize (Zea mays) seeds are the major source of starch all over the world and the excellent model for researching starch synthesis. Seed starch content is a typical quantitative phenotype and many reports revealed that the glycolytic enzymes are involved in regulating starch synthesis, however the regulatory mechanism is still unclear. Here, we present a comparative phosphoproteomic study of three maize inbred lines with different seed starch content. It reveals that abundances of 62 proteins and 63 phosphoproteins were regulated during maize seed development. Dynamics of 17 enzymes related to glycolysis and starch synthesis were used to construct a phosphorylation regulatory network of starch synthesis. It shows that starch synthesis and glycolysis in maize seeds utilize the same hexose phosphates pool coming from sorbitol and sucrose as carbon source, and phosphorylation of ZmENO1 are suggested to contribute to increase starch content, because it is positively related to seed starch content in different developmental stages and different lines, and the phosphor-mimic mutant (ZmENO1S43D) damaged its enzyme activity which is vital in glycolysis. Our results provide a new sight into regulatory process of seed starch synthesis and can be used in maize breeding for high starch content.

Identification of Chaetocin as a Potent non-ROS-mediated Anticancer Drug Candidate for Gastric Cancer.

Chaetocin, a natural product extracted from Chaetomium species, possesses anticancer effects in several kinds of tumors. However, it remains unclear whether the potential indication for chaetocin could also include human gastric cancer. We found here that chaetocin induced caspase-dependent and -independent apoptosis in human gastric cancer cell lines, which greatly depended on BID-mediated AIF translocation. Despite not increasing the intercellular ROS levels in gastric cancer cells, chaetocin did cause a reduction in mitochondrial membrane potential probably through its regulation on the expression of Bcl-2 and BAX. Chaetocin could also induce autophagy in gastric cancer cells; blocking autophagy by chloroquine enhanced the cytotoxicity of chaetocin. Chaetocin was further found to suppress the growth of gastric cancer xenograft in nude mice. Therefore, our study provides first evidence that chaetocin has an anticancer efficacy against gastric cancer and the combined use of chaetocin with autophagy inhibitors may enhance the therapeutic effect for gastric cancer. As chronic and exorbitant ROS levels instigate drug resistance, chaetocin, which eradicates gastric cancer cells without increasing ROS levels, may initiate a new line of non-ROS-mediated anti-tumor strategy.

Resistance mechanisms and population structure of highly drug resistant Klebsiella in Pakistan during the introduction of the carbapenemase NDM-1.

Klebsiella pneumoniae is a major threat to public health with the emergence of isolates resistant to most, if not all, useful antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae collected from patients resident in a region of Pakistan, during the period 2010-2012, when the now globally-distributed carbapenemase bla-NDM-1 was being acquired by Klebsiella. We observed two dominant lineages, but neither the overall resistance profile nor virulence-associated factors, explain their evolutionary success. Phenotypic analysis of resistance shows few differences between the acquisition of resistance genes and the phenotypic resistance profile, including beta-lactam antibiotics that were used to treat ESBL-positive strains. Resistance against these drugs could be explained by inhibitor-resistant beta-lactamase enzymes, carbapenemases or ampC type beta-lactamases, at least one of which was detected in most, but not all relevant strains analysed. Complete genomes for six selected strains are reported, these provide detailed insights into the mobile elements present in these isolates during the initial spread of NDM-1. The unexplained success of some lineages within this pool of highly resistant strains, and the discontinuity between phenotypic resistance and genotype at the macro level, indicate that intrinsic mechanisms contribute to competitive advantage and/or resistance.

High expression of Tob1 indicates poor survival outcome and promotes tumour progression via a Wnt positive feedback loop in colon cancer.

Tob1, a Tob/BTG anti-proliferative protein family member, functions as a tumour suppressor in many cancers. Here, we reveal a unique oncogenic role of Tob1 in colon cancer. Tob1 expression was upregulated during colon cancer progression, was significantly correlated with tumour size and tumour differentiation, and was a prognostic indicator of colon cancer. Unlike in other cancers, where nuclear Tob1 performs anticancer activity, Tob1 is predominantly localized in the cytosol of colon cancer cells, where this protein binds and stabilizes ß-catenin to activate Wnt/ß-catenin signalling, which in turn enhances Tob1 expression, thus forming a positive feedback loop to promote cell proliferation. Moreover, Tob1 deficiency led to reduced tumourigenesis in AOM/DSS-treated and ApcMin/+ mice. Our findings provide important insights into a previously unrecognized oncogenic role of Tob1 in colon cancer and suggest that Tob1 is an adverse prognostic factor and therapeutic target for colon cancer.

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.

Berberine decelerates glucose metabolism via suppression of mTOR­dependent HIF­1α protein synthesis in colon cancer cells.

Hyperactivated glucose uptake and glycolytic metabolism are considered as a hallmark of cancer. Berberine, a natural alkaloid with tumor­selective anticancer effects, has been shown to promote glucose uptake in metabolic tissues and cells. However, whether and how berberine regulates the glucose metabolism of cancer cells are still poorly understood. In the present study, we revealed that berberine, which suppressed the growth of colon cancer cell lines HCT116 and KM12C, greatly inhibited the glucose uptake and the transcription of glucose metabolic genes, GLUT1, LDHA and HK2 in these two cell lines as assessed by RT­qPCR. A mechanistic study further indicated that the protein expression but not mRNA transcription of HIF­1α, a well­known transcription factor critical for dysregulated cancer cell glucose metabolism, was dramatically inhibited in berberine­treated colon cancer cell lines. Using western blot analysis, this regulation appears to occur via protein synthesis but not protein stability as blockade of HIF­1α protein degradation by hypoxia mimic desferrioxamine (DFX) or proteasome inhibitor MG132 did not affect berberine's effect. In addition, mTOR signaling previously reported to regulate HIF­1α protein synthesis was further found to be suppressed by berberine. Taken together, our results indicated that berberine inhibits overactive glucose metabolism of colon cancer cells via suppressing mTOR­depended HIF­1α protein synthesis, which provided not only a novel mechanism involved in berberine's tumor­specific toxicity but also a theoretical basis for the development of berberine for colon cancer treatment.

Mucosal-Associated Invariant T Cells Augment Immunopathology and Gastritis in Chronic Helicobacter pylori Infection.

Mucosal-associated invariant T (MAIT) cells produce inflammatory cytokines and cytotoxic granzymes in response to by-products of microbial riboflavin synthesis. Although MAIT cells are protective against some pathogens, we reasoned that they might contribute to pathology in chronic bacterial infection. We observed MAIT cells in proximity to Helicobacter pylori bacteria in human gastric tissue, and so, using MR1-tetramers, we examined whether MAIT cells contribute to chronic gastritis in a mouse H. pylori SS1 infection model. Following infection, MAIT cells accumulated to high numbers in the gastric mucosa of wild-type C57BL/6 mice, and this was even more pronounced in MAIT TCR transgenic mice or in C57BL/6 mice where MAIT cells were preprimed by Ag exposure or prior infection. Gastric MAIT cells possessed an effector memory Tc1/Tc17 phenotype, and were associated with accelerated gastritis characterized by augmented recruitment of neutrophils, macrophages, dendritic cells, eosinophils, and non-MAIT T cells and by marked gastric atrophy. Similarly treated MR1-/- mice, which lack MAIT cells, showed significantly less gastric pathology. Thus, we demonstrate the pathogenic potential of MAIT cells in Helicobacter-associated immunopathology, with implications for other chronic bacterial infections.

Phylogenetic Analysis of Klebsiella pneumoniae from Hospitalized Children, Pakistan.

Klebsiella pneumoniae shows increasing emergence of multidrug-resistant lineages, including strains resistant to all available antimicrobial drugs. We conducted whole-genome sequencing of 178 highly drug-resistant isolates from a tertiary hospital in Lahore, Pakistan. Phylogenetic analyses to place these isolates into global context demonstrate the expansion of multiple independent lineages, including K. quasipneumoniae.

Berberine inhibits colitis-associated tumorigenesis via suppressing inflammatory responses and the consequent EGFR signaling-involved tumor cell growth.

The anti-inflammatory and anti-tumor effects of berberine, a traditional Chinese medicine, were separately discovered in pathological intestinal tissues. However, whether the anti-inflammatory effect of berberine contributes to its anti-tumor effect on colitis-associated colorectal cancer (CACRC) remains unknown. In the present study, we found that berberine effectively inhibited colitis-associated tumorigenesis and colonic epithelium hyperproliferation in dextran sulfate sodium (DSS)-treated ApcMin/+ mice. A mechanistic study identified that these inhibitory effects of berberine occurred through blocking interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) expression in colonic macrophages. An in vitro study on cell lines identified that berberine treatment of Raw 264.7 macrophages resulted in conditioned media with fewer proliferative effects on a cell line with a heterozygous Apc mutation (Immorto-Min colonic epithelium, IMCE). EGFR-ERK signaling act downstream of berberine/pro-inflammatory cytokines axis to regulate CACRC cell proliferation. Furthermore, in vivo administration of IL-6 to DSS-treated ApcMin/+ mice effectively weakened the inhibitory effects of berberine on tumorigenesis and EGFR-ERK signaling in colon tissues. Altogether, the results of our studies have revealed that berberine inhibits the development of CACRC by interfering with inflammatory response-driven EGFR signaling in tumor cell growth. The findings of this study support the possibility that berberine and other anti-inflammatory drugs may be beneficial in the treatment of CACRC.

Exo70 is an independent prognostic factor in colon cancer.

Exo70, a key component of the Exocyst complex, plays important roles in human cancer progression beyond exocytosis. However, the expression of Exo70 and its prognostic value for patients with colon cancer has not been well investigated to date. In this study, we observed that the mRNA and protein levels of Exo70 were upregulated in 11 of 13 colon cancer tissues, compared with their normal counterparts, which was validated by immunohistochemical analysis in a tissue microarray containing 89 pairs of colon cancer tissues and the matched adjacent normal tissues. Statistical analysis revealed that Exo70 expression is positively correlated with tumor size, invasion depth, TNM stage and distant metastasis. Kaplan-Meier survival analysis showed that colon cancer patients with higher Exo70 expression have a poorer clinical outcome than those with lower Exo70 expression. Multivariate Cox regression analysis revealed that Exo70, age and distant metastasis were there independent prognostic factors for overall survival rate of colon cancer patients. Through gain- and loss of Exo70 in colon cancer cells, we found that Exo70 could enhance the migration ability of colon cancer cells. Taken together, our studies revealed that Exo70 might be a promising negative prognostic factor and a potential therapeutic target for colon cancer.