Targeting RLIP with CRISPR/Cas9 controls tumor growth.

Breast cancer (BC) remains one of the major causes of cancer deaths in women. Over half of all BCs carry genetic defects in the gene encoding p53, a powerful tumor suppressor. P53 is known as the 'guardian of the genome' because it is essential for regulating cell division and preventing tumor formation. Ral-interacting protein (RLIP) is a modular protein capable of participating in many cellular functions. Blocking this stress-responsive protein, which is overexpressed during malignancy, enables BC cells to overcome the deleterious effects of p53 loss more effectively. In the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas9) system, a single-guide RNA (sgRNA) recognizes a specific DNA sequence and directs the endonuclease Cas9 to make a double-strand break, which enables editing of targeted genes. Here, we harnessed CRISPR/Cas9 technology to target the RLIP gene in BC cells. We screened sgRNAs using a reporter system and lentivirally delivered them, along with Cas9, to BC cells for validation. We then assessed the survival, proliferation, and tumorigenicity of BC cells in vitro and the growth of tumors in vivo after CRISPR-mediated knockdown of RLIP. Doxycycline-inducible expression of Cas9 in BC cells transduced with lentiviral vectors encoding the sgRNAs disrupted the RLIP gene, leading to inhibition of BC cell proliferation both in vitro and in vivo, with resected tumors showing reduced levels of the survival and proliferation markers Ki67, RLIP, pAkt, and survivin, the cell cycle protein CDK4, and the mesenchymal marker vimentin, as well as elevated levels of the differentiation protein E-cadherin and pro-apoptotic protein Bim. Inducible Cas9/sgRNA-transduced BC cells without doxycycline treatment did not exhibit altered cell survival or proliferation in vitro or in vivo. Our study provides proof-of-concept that the CRISPR/Cas9 system can be utilized to target RLIP in vitro and in vivo.

RLIP inhibition suppresses breast-to-lung metastasis.

Breast tumor metastasis is a leading cause of cancer-related deaths worldwide. Breast cancer (BC) cells frequently metastasize to the lungs, where they pose a formidable therapeutic challenge. In the current study, we evaluated the anti-proliferative and anti-metastatic effects of 2'-hydroxyflavanone (2HF) and RLIP inhibition in an array of triple-negative BC cell lines and an orthotopic mouse model of breast-to-lung metastasis. Compared to control treatment, RLIP inhibition reduced in-vitro cell viability and suppressed the migratory and invasive potential of BC cells. In-vitro studies showed that 2HF treatment reduced the expression of RLIP, KRAS, pERK, pSTAT3, and pP70S6K. Further, mice orthotopically implanted with lung-seeking luciferase-expressing TMD231 cells were treated with 2HF (50 mg/kg, b.w.), RLIP antisense (RAS; 5 mg/kg, b.w.), RLIP antibody (Rab; 5 mg/kg, b.w.) or a combination of 2HF + RAS + Rab. 2HF-, RAS-, and Rab-treated mice exhibited significantly lower primary tumor weight and reduced lung metastasis compared to control mice. Mice treated with a combination of 2HF + RAS + Rab exhibited no metastasis and significantly lower tumor weight than the single agent-treated mice. Collectively, our results suggest that 2HF has potential to be combined with RLIP inhibition/depletion to more effectively suppress primary breast tumor growth and metastasis to the lungs.

2'-Hydroxyflavanone induced changes in the proteomic profile of breast cancer cells.

In spite of rapid advances in understanding of signaling networks associated with the incidence and therapeutic-sensitivity, breast cancer (BC) still remains the most commonly diagnosed and prevalent cancer in women. Emergence of resistance to hormonal interventions in estrogen-receptor (ER) positive BC coupled to loss of ER expression and activation of ER-independent growth factor, heat-shock, MYC and WNT pathways along with distinct mechanisms of therapeutic-resistance in HER2 over-expressing and triple-negative subtypes of BC collectively necessitates deeper profiling of the mechanistic networks regulated by potential lead anticancer compounds intended for further development to target BC. A significant part of the search for novel lead anticancer compounds for BC has focused on phytochemicals including flavonoids found in citrus fruits, which have shown promising anticancer activity. Based on the initial studies which revealed the anticancer effect of 2HF in BC, we employed an advanced TMT 10plex labeled proteomic approach to characterize the changes in non-phosphorylated and phosphorylated proteomic profile of ER+ MCF7, triple-negative MDA-MB231 and HER2+ SKBR3 BC cells, and MCF10A normal breast epithelial cells. 2HF induced significant changes in the proteins responsible for BC incidence, metastases and therapeutic sensitivity in BC cells.

2'-Hydroxyflavanone inhibits in vitro and in vivo growth of breast cancer cells by targeting RLIP76.

Consumption of citrus-fruits is associated with reduced incidence of breast cancer (BC), the most common cancer diagnosed in women across the globe. In this study, we investigated the anticancer potential of 2-Hydroxyflavanone (2HF) in BC. 2HF, a citrus-bioflavonoid, has demonstrated anticancer properties in various cancers, but its anticancer role in BC has not been well studied. We investigated the in vitro and in vivo growth inhibitory effects of 2HF in an array of BC lines and in xenograft mouse models of ER-positive and HER2-positive BC cells. Compared to control, 2HF treatment reduced cell viability and suppressed migratory and invasive potential of BC cells, while, no growth inhibitory effects were observed in non-tumorigenic breast epithelial cells. Further, 2HF inhibited the expression of RLIP76, a stress-defensive and anti-apoptotic protein, which is over-expressed in BC cells and simultaneously reduced proliferation of BC cells. Nude mice bearing MCF7 or SKBR3 BC cells xenografts treated with either 2HF or targeting RLIP76 by RLIP76-antisense or RLIP76-antibody treatment had significantly lower tumor-weight as compared to corresponding controls. In addition, Western-blotting and immunohistochemical analysis of tumor tissue from control and treatment group mice showed that 2HF decreased protein expression levels of RLIP76, and the decrease was similar to those seen following RLIP76-antisense treatment. Furthermore, 2HF decreased expression of Ki67, CD31, vimentin, inhibited phosphorylation of Akt and expression of survivin and Bcl2, and increased levels of Bax, E-cadherin, and cleaved-PARP. Therefore, our results indicate that 2HF may suppress BC growth in vitro and in vivo by targeting RLIP76, and may serve as a potential adjuvant treatment in BC patients.

Piperlongumine potentiates the effects of gemcitabine in in vitro and in vivo human pancreatic cancer models.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers due to a late diagnosis and poor response to available treatments. There is a need to identify complementary treatment strategies that will enhance the efficacy and reduce the toxicity of currently used therapeutic approaches. We investigated the ability of a known ROS inducer, piperlongumine (PL), to complement the modest anti-cancer effects of the approved chemotherapeutic agent gemcitabine (GEM) in PDAC cells in vitro and in vivo. PDAC cells treated with PL + GEM showed reduced cell viability, clonogenic survival, and growth on Matrigel compared to control and individually-treated cells. Nude mice bearing orthotopically implanted MIA PaCa-2 cells treated with both PL (5 mg/kg) and GEM (25 mg/kg) had significantly lower tumor weight and volume compared to control and single agent-treated mice. RNA sequencing (RNA-Seq) revealed that PL + GEM resulted in significant changes in p53-responsive genes that play a role in cell death, cell cycle, oxidative stress, and DNA repair pathways. Cell culture assays confirmed PL + GEM results in elevated ROS levels, arrests the cell cycle in the G0/G1 phase, and induces PDAC cell death. We propose a mechanism for the complementary anti-tumor effects of PL and GEM in PDAC cells through elevation of ROS and transcription of cell cycle arrest and cell death-associated genes. Collectively, our results suggest that PL has potential to be combined with GEM to more effectively treat PDAC.

Flaxseed Consumption Inhibits Chemically Induced Lung Tumorigenesis and Modulates Expression of Phase II Enzymes and Inflammatory Cytokines in A/J Mice.

Flaxseed consumption is associated with reduced oxidative stress and inflammation in lung injury models and has shown anticancer effects for breast and prostate tissues. However, the chemopreventive potential of flaxseed remains unexplored for lung cancer. In this study, we investigated the effect of flaxseed on tobacco smoke carcinogen (NNK)-induced lung tumorigenesis in an A/J mouse model. Mice exposed to NNK were fed a control diet or a 10% flaxseed-supplemented diet for 26 weeks. Flaxseed-fed mice showed reduced lung tumor incidence (78%) and multiplicity, with an average of 2.7 ± 2.3 surface lung tumor nodules and 1.0 ± 0.9 H&E cross-section nodules per lung compared with the control group, which had 100% tumor incidence and an average of 10.2 ± 5.7 surface lung tumor nodules and 3.9 ± 2.6 H&E cross-section nodules per lung. Furthermore, flaxseed-fed mice had a lower incidence of adenocarcinomas compared with control-fed mice. Western blotting performed on normal lung tissues showed flaxseed suppressed phosphorylation (activation) of p-AKT, p-ERK, and p-JNK kinases. RNA-Seq data obtained from normal lung and lung tumors of control and flaxseed-fed mice suggested that flaxseed intake resulted in differential expression of genes involved in inflammation-mediated cytokine signaling (IL1, 6, 8, 9, and 12α), xenobiotic metabolism (several CYPs, GSTs, and UGTs), and signaling pathways (AKT and MAPK) involved in tumor cell proliferation. Together, our results indicate that dietary flaxseed supplementation may be an effective chemoprevention strategy for chemically induced lung carcinogenesis by altering signaling pathways, inflammation, and oxidative stress. Cancer Prev Res; 11(1); 27-37. ©2017 AACR.

Oxidative stress and dietary phytochemicals: Role in cancer chemoprevention and treatment.

Several epidemiological observations have shown an inverse relation between consumption of plant-based foods, rich in phytochemicals, and incidence of cancer. Phytochemicals, secondary plant metabolites, via their antioxidant property play a key role in cancer chemoprevention by suppressing oxidative stress-induced DNA damage. In addition, they modulate several oxidative stress-mediated signaling pathways through their anti-oxidant effects, and ultimately protect cells from undergoing molecular changes that trigger carcinogenesis. In several instances, however, the pro-oxidant property of these phytochemicals has been observed with respect to cancer treatment. Further, in vitro and in vivo studies show that several phytochemicals potentiate the efficacy of chemotherapeutic agents by exacerbating oxidative stress in cancer cells. Therefore, we reviewed multiple studies investigating the role of dietary phytochemicals such as, curcumin (turmeric), epigallocatechin gallate (EGCG; green tea), resveratrol (grapes), phenethyl isothiocyanate (PEITC), sulforaphane (cruciferous vegetables), hesperidin, quercetin and 2'-hydroxyflavanone (2HF; citrus fruits) in regulating oxidative stress and associated signaling pathways in the context of cancer chemoprevention and treatment.

Translational opportunities for broad-spectrum natural phytochemicals and targeted agent combinations in breast cancer.

Breast cancer (BC) prevention and therapy in the context of life-style risk factors and biological drivers is a major focus of developmental therapeutics in oncology. Obesity, alcohol, chronic estrogen signaling and smoking have distinct BC precipitating and facilitating effects that may act alone or in combination. A spectrum of signaling events including enhanced oxidative stress and changes in estrogen-receptor (ER)-dependent and -independent signaling drive the progression of BC. Breast tumors modulate ERα/ERß ratio, upregulate proliferative pathways driven by ERα and HER2 with a parallel loss and/or downregulation of tumor suppressors such as TP53 and PTEN which together impact the efficacy of therapeutic strategies and frequently lead to emergence of drug resistance. Natural phytochemicals modulate oxidative stress, leptin, integrin, HER2, MAPK, ERK, Wnt/ß-catenin and NFκB signaling along with regulating ERα and ERß, thereby presenting unique opportunities for both primary and combinatorial interventions in BC. In this regard, this article focuses on critical analyses of the evidence from multiple studies on the efficacy of natural phytochemicals in BC. In addition, areas in which the combinations of such effective natural phytochemicals with approved and/or developing anticancer agents can be translationally beneficial are discussed to derive evidence-based inference for addressing challenges in BC control and therapy.

2'-Hydroxyflavanone: A novel strategy for targeting breast cancer.

Breast cancer is the most common cancer in women that is driven by cross-talk with hormonal and cellular signaling pathways. The natural phytochemicals, due to broad-spectrum anti-inflammatory and anti-cancerous properties, present with novel opportunities for targeting breast cancer. Intake of citrus fruits is known to reduce the risk for incidence of breast cancer. Hence, we tested the efficacy of citrus flavonoid 2'-hydroxyflavanone (2HF) in breast cancer. 2HF inhibited survival, clonogenic ability, cell cycle progression and induced apoptosis in breast cancer cells. 2HF also decreased VEGF levels and inhibited migratory capacity of breast cancer cells. Administration of 2HF led to regression of triple-negative MDA-MB-231 tumors in the mice xenograft model. 2HF decreased the levels of RLIP76 both in vitro studies and in vivo MDA-MB-231 xenograft model of breast cancer. Western blot and histopathological analyses of resected tumors showed a decline in the levels of survival and proliferation markers Ki67, pAkt, survivin, and cell cycle proteins CDK4 and cyclin B1. 2HF treatment led to inhibition of angiogenesis as determined by decreased VEGF levels in vitro and angiogenesis marker CD31 in vivo. 2HF reversed the pro-/anti-apoptotic ratio of BAX/BCL-2 by decreasing anti-apoptotic protein BCL-2 and increasing pro-apoptotic proteins BAX and BIM in vivo. 2HF also decreased the mesenchymal markers vimentin and fibronectin along with causing a parallel increase in pro-differentiation protein E-cadherin. Collectively, the ability of 2HF to decrease RLIP76, VEGF and regulate critical proliferative, apoptotic and differentiation proteins together provides strong rationale to further develop 2HF based interventions for targeting breast cancer.

Enterolactone Induces G1-phase Cell Cycle Arrest in Nonsmall Cell Lung Cancer Cells by Downregulating Cyclins and Cyclin-dependent Kinases.

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anticancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study, we investigated the anticancer effects of EL for several nonsmall cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The antiproliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G1-phase cell cycle arrest. Molecular studies revealed that EL decreased mRNA or protein expression levels of the G1-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21WAF1/CIP1, a negative regulator of the G1 phase. The results suggest that EL inhibits the growth of NSCLC cell lines by downregulating G1-phase cyclins and CDKs, and upregulating p21WAF1/CIP1, which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.

Enterolactone alters FAK-Src signaling and suppresses migration and invasion of lung cancer cell lines.

BACKGROUND: Systemic toxicity of chemotherapeutic agents and the challenges associated with targeting metastatic tumors are limiting factors for current lung cancer therapeutic approaches. To address these issues, plant-derived bioactive components have been investigated for their anti-cancer properties because many of these agents are non-toxic to healthy tissues. Enterolactone (EL) is a flaxseed-derived mammalian lignan that has demonstrated anti-migratory properties for various cancers, but EL has not been investigated in the context of lung cancer, and its anticancer mechanisms are ill-defined. We hypothesized that EL could inhibit lung cancer cell motility by affecting the FAK-Src signaling pathway. METHODS: Non-toxic concentrations of EL were identified for A549 and H460 human lung cancer cells by conducting 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Dephenyltetrazolium Bromide (MTT) assays. The anti-migratory and anti-invasive potential of EL for lung cancer cell lines was determined by scratch wound healing and Matrigel® invasion assays. Changes in filamentous actin (F-actin) fiber density and length in EL-treated cells were determined using phalloidin-conjugated rhodamine dye and fluorescent microscopy. Vinculin expression in focal adhesions upon EL treatment was determined by immunocytochemistry. Gene and protein expression levels of FAK-Src signaling molecules in EL-treated lung cancer cells were determined using PCR arrays, qRT-PCR, and western blotting. RESULTS: Non-toxic concentrations of EL inhibited lung cancer cell migration and invasion in a concentration- and time-dependent manner. EL treatment reduced the density and number of F-actin fibers in lung cancer cell lines, and reduced the number and size of focal adhesions. EL decreased phosphorylation of FAK and its downstream targets, Src, paxillin, and decreased mRNA expression of cell motility-related genes, RhoA, Rac1, and Cdc42 in lung cancer cells. CONCLUSIONS: Our data suggest that EL suppresses lung cancer cell motility and invasion by altering FAK activity and subsequent activation of downstream proteins needed for focal adhesion formation and cytoskeletal rearrangement. Therefore, administration of EL may serve as a safe and complementary approach for inhibiting lung tumor cell motility, invasion, and metastasis.

Piperlongumine induces pancreatic cancer cell death by enhancing reactive oxygen species and DNA damage.

Pancreatic cancer is one of the most deadly cancers with a nearly 95% mortality rate. The poor response of pancreatic cancer to currently available therapies and the extremely low survival rate of pancreatic cancer patients point to a critical need for alternative therapeutic strategies. The use of reactive oxygen species (ROS)-inducing agents has emerged as an innovative and effective strategy to treat various cancers. In this study, we investigated the potential of a known ROS inducer, piperlongumine (PPLGM), a bioactive agent found in long peppers, to induce pancreatic cancer cell death in cell culture and animal models. We found that PPLGM inhibited the growth of pancreatic cancer cell cultures by elevating ROS levels and causing DNA damage. PPLGM-induced DNA damage and pancreatic cancer cell death was reversed by treating the cells with an exogenous antioxidant. Similar to the in vitro studies, PPLGM caused a reduction in tumor growth in a xenograft mouse model of human pancreatic cancer. Tumors from the PPLGM-treated animals showed decreased Ki-67 and increased 8-OHdG expression, suggesting PPLGM inhibited tumor cell proliferation and enhanced oxidative stress. Taken together, our results show that PPLGM is an effective inhibitor for in vitro and in vivo growth of pancreatic cancer cells, and that it works through a ROS-mediated DNA damage pathway. These findings suggest that PPLGM has the potential to be used for treatment of pancreatic cancer.

Overexpression of peptide deformylase in breast, colon, and lung cancers.

BACKGROUND: Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. METHODS: The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. RESULTS: PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. CONCLUSIONS: This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation.

Notch signaling: a hero or villain in the war against cancer?

The Notch signal transduction pathway regulates cell fate decisions throughout embryonic development. The mechanisms through which Notch signaling maintains cellular integrity are well understood. However, Notch signaling is more complex than previously thought as Notch is also involved in cancer where it functions as both an oncogene and tumor suppressor depending on the cellular context. Aberrant activation of oncogenic Notch is found in various cancers prompting the search for therapeutic agents to attenuate constitutively active Notch. However, there is also substantial evidence that Notch signaling suppresses tumor growth and progression, suggesting that Notch activators might be of therapeutic benefit in other cancers. This editorial describes the dual role of Notch signaling observed within and across multiple cancers. We highlight a study in non-small cell lung cancer cells (NSCLC) revealing a tumor suppressive role for endothelial cell Dll4-activated Notch1 and the underlying molecular mechanism involving suppression of PI3K signaling.

Syntenic relationships among legumes revealed using a gene-based genetic linkage map of common bean (Phaseolus vulgaris L.).

Molecular linkage maps are an important tool for gene discovery and cloning, crop improvement, further genetic studies, studies on diversity and evolutionary history, and cross-species comparisons. Linkage maps differ in both the type of marker and type of population used. In this study, gene-based markers were used for mapping in a recombinant inbred (RI) population of Phaseolus vulgaris L. P. vulgaris, common dry bean, is an important food source, economic product, and model organism for the legumes. Gene-based markers were developed that corresponded to genes controlling mutant phenotypes in Arabidopsis thaliana, genes undergoing selection during domestication in maize, and genes that function in a biochemical pathway in A. thaliana. Sequence information, including introns and 3' UTR, was generated for over 550 genes in the two genotypes of P. vulgaris. Over 1,800 single nucleotide polymorphisms and indels were found, 300 of which were screened in the RI population. The resulting LOD 2.0 map is 1,545 cM in length and consists of 275 gene-based and previously mapped core markers. An additional 153 markers that mapped at LOD <1.0 were placed in genetic bins. By screening the parents of other mapping populations, it was determined that the markers were useful for other common Mesoamerican × Andean mapping populations. The location of the mapped genes relative to their homologs in Arabidopsis thaliana (At), Medicago truncatula (Mt), and Lotus japonicus (Lj) were determine by using a tblastx analysis with the current psedouchromosome builds for each of the species. While only short blocks of synteny were observed with At, large-scale macrosyntenic blocks were observed with Mt and Lj. By using Mt and Lj as bridging species, the syntenic relationship between the common bean and peanut was inferred.

Synteny mapping between common bean and soybean reveals extensive blocks of shared loci.

BACKGROUND: Understanding syntentic relationship between two species is critical to assessing the potential for comparative genomic analysis. Common bean (Phaseolus vulgaris L.) and soybean (Glycine max L.), the two most important members of the Phaseoleae legumes, appear to have a diploid and polyploidy recent past, respectively. Determining the syntentic relationship between these two species will allow researchers to leverage not only genomic resources but also genetic data for important agronomic traits to improve both of these species. RESULTS: Genetically-positioned transcript loci of common bean were mapped relative to the recent soybean 1.01 pseudochromosome assembly. In nearly every case, each common bean locus mapped to two loci in soybean, a result consistent with the duplicate polyploidy history of soybean. Blocks of synteny averaging 32 cM in common bean and 4.9 Mb in soybean were observed for all 11 common bean linkage groups, and these blocks mapped to all 20 soybean pseudochromosomes. The median physical-to-genetic distance ratio in common bean (based on soybean physical distances) was approximately 120 kb/cM. approximately 15,000 common bean sequences (primarily EST contigs and EST singletons) were electronically positioned onto the common bean map using the shared syntentic blocks as references points. CONCLUSION: The collected evidence from this mapping strongly supports the duplicate history of soybean. It further provides evidence that the soybean genome was fractionated and reassembled at some point following the duplication event. These well mapped syntentic relationships between common bean and soybean will enable researchers to target specific genomic regions to discover genes or loci that affect phenotypic expression in both species.