Posts Tagged ‘PI3K’

Mutations in RAS genes

Larry H Bernstein, MD, FCAP, Curator



Novel Mechanism Targets “Undruggable” RAS Oncogenes

Dr. Reddy discussing findings from new research on developing drugs toward RAS.

Mechanism of new research targeting previously undruggable RAS gene. [Cell, Volume 165, 21 April 2016]


For greater than 30% of human cancers, RAS genes are mutated and have been implicated as key tumor drivers—making them some of the most sought after cancer drug targets. However, the absence of any drug-binding pockets in the mutant RAS proteins has made drug development extremely difficult. Yet now, a new study from researchers at the Icahn School of Medicine at Mount Sinai has identified a new mechanism for targeting this important cancer gene.

Mutations in RAS genes—such as HRAS, KRAS, and NRAS—are frequently observed in many of the most common and lethal tumors, including cancers of the pancreas, lung, and colon. While scientists have made significant headway in understanding these mutations and their impact on cellular signaling, little headway has been made toward developing drugs that systematically target the RAS oncogenes. This lack of progress has led many in the field to label RAS an “undruggable” cancer gene.

The Mt. Sinai team identified what they believe to be the first small molecule able to simultaneously inhibit the different signaling pathways activated by RAS oncogenes. This compound called rigosertib or ON01910.Na, acts as a protein-protein interaction inhibitor that prevents binding between RAS and signaling proteins (including RAF, PI3K, and others) that drive a cell into a cancer cell.

“Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway,” the authors wrote. “We also find that rigosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.”

The findings from this study were published recently in Cell through an article entitled “A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling.”

Additionally, the investigators performed structural experiments to confirm the mode of action for rigosertib and also demonstrated the potential for this targeted mechanism in the treatment of several RAS-driven cancers.

“This discovery is a significant breakthrough for the cancer field,” explained senior study author E. Premkumar Reddy, Ph.D., professor of oncological sciences at the Icahn School of Medicine at Mount Sinai. “Rigosertib’s mechanism of action represents a new paradigm for attacking the intractable RAS oncogenes. Our current focus is to use the information from our studies with rigosertib to design the next generation of small molecule RAS-targeting therapies, and we are excited to have recently identified several compounds which we think improve on the qualities of rigosertib.”


A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling

Sai Krishna Athuluri-Divakar, Rodrigo Vasquez-Del Carpio,….., Aneel K. Aggarwal, E. Premkumar Reddycorrespondence

  • Rigosertib binds to the RAS-binding domains (RBDs) of multiple RAS effectors
  • Binding of rigosertib to RAF-RBD inhibits RAS-RAF interaction and impairs the kinase
  • Rigosertib inhibits MEK-ERK pathway activated by growth factors and oncogenic RAS

Oncogenic activation of RAS genes via point mutations occurs in 20%–30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.


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Curator: Ritu Saxena, Ph.D.


Melanoma represents approximately 4% of human skin cancers, yet accounts for approximately 80% of deaths from cutaneous neoplasms. It remains one of the most common types of cancer among young adults. Melanoma is recognized as the most common fatal skin cancer with its incidence rising to 15 fold in the past 40 years in the United States. Melanoma develops from the malignant transformation of melanocytes, the pigment-producing cells that reside in the basal epidermal layer in human skin. (Greenlee RT, et al, Cancer J Clin. Jan-Feb 2001;51(1):15-36 ; Weinstock MA, et al, Med Health R I. Jul 2001;84(7):234-6).  Classic clinical signs of melanoma include change in color, recent enlargement, nodularity, irregular borders, and bleeding. Cardinal signs of melanoma are sometimes referred to by the mnemonic ABCDEs (asymmetry, border irregularity, color, diameter, elevation) (Chudnovsky Y, et al. J Clin Invest, 1 April 2005; 115(4): 813–824).

Clinical characteristics

Melanoma primarily affects fair-haired and fair-skinned individuals, and those who burn easily or have a history of severe sunburn are at higher risk than their darkly pigmented, age-matched controls. The exact mechanism and wavelengths of UV light that are the most critical remain controversial, but both UV-A (wavelength 320–400 nm) and UV-B (290–320 nm) have been implicated (Jhappan C, et al, Oncogene, 19 May 2003;22(20):3099-112). Case-control studies have identified several risk factors in populations susceptible to developing melanoma. MacKie RM et al (1989) stated that the relative risk of cutaneous melanoma is estimated from the four strongest risk factors identified by conditional logistic regression. These factors are

  • total number of benign pigmented naevi above 2 mm diameter;
  • freckling tendency;
  • number of clinically atypical naevi (over 5 mm diameter and having an irregular edge, irregular pigmentation, or inflammation); and
  • a history of severe sunburn at any time in life.

Use of this risk-factor chart should enable preventive advice for and surveillance of those at greatest risk (MacKie RM, et al, Lancet 26 Aug1989;2(8661):487-90).

Cutaneous melanoma can be subdivided into several subtypes, primarily based on anatomic location and patterns of growth (Table 1).


Table 1: Clinical Classification of Melanoma (Chudnovsky Y, et al, 2005)

The genetics of melanoma

As in many cancers, both genetic predisposition and exposure to environmental agents are risk factors for melanoma development. Many studies conducted over several decades on benign and malignant melanocytic lesions as well as melanoma cell lines have implicated numerous genes in melanoma development and progression.


Table 2: Genes involved in Melanoma (Chudnovsky Y, et al, 2005)

Apart from the risk factors such as skin pigmentation, freckling, and so on, another significant risk factor is ‘strong family history of melanoma’. Older case-control studies of patients with familial atypical mole-melanoma (FAMM) syndrome suggested an elevated risk of ∼434-to 1000-fold over the general population (Greene MH, et al, Ann Intern Med, Apr 1985;102(4):458-65). A more recent meta-analysis of family history found that the presence of at least one first-degree relative with melanoma increases the risk by 2.24-fold (Gandini S, et al, Eur J Cancer, Sep 2005;41(14):2040-59). Genetic studies of melanoma-prone families have given important clues regarding melanoma susceptibility loci.

CDKN2A, the familial melanoma locus

CDKN2A is located at chromosome 9p21 and is composed of 4 exons (E) – 1α, 1β, 2, and 3. LOH or mutations at this locus cosegregated with melanoma susceptibility in familial melanoma kindred and 9p21 mutations have been observed in different cancer cell lines. The locus encodes two tumor suppressors via alternate reading frames, INK4 (p16INK4a) and ARF (p14ARF). INK4A and ARF encode alternative first exons, 1α and 1β respectively and different promoters. INK4A is translated from the splice product of E1α, E2, and E3, while ARF is translated from the splice product of E1β, E2, and E3. Second exons of the two proteins are shared and two translated proteins share no amino acid homology.

INK4A is the founding member of the INK4 (Inhibitor of cyclin-dependent kinase 4) family of proteins and inhibits the G1 cyclin-dependent kinases (CDKs) 4/6, which phosphorylate and inactivate the retinoblastoma protein (RB), thereby allowing for S-phase entry. Thus, loss of INK4K function promotes RB inactivation through hyperphosphorylation, resulting in unconstrained cell cycle progression.

ARF (Alternative Reading Frame) protein of the locus inhibits HDM2-mediated ubiquitination and subsequent degradation of p53. Thus, loss of ARF inactivates another tumor suppressor, p53. The loss of p53 impairs mechanisms that normally target genetically damaged cells for cell cycle arrest and/or apoptosis, which leads to proliferation of damaged cells. Loss of CDKN2A therefore contributes to tumorigenesis by disruption of both the pRB and p53 pathways.

figure 1

Figure 1:  Genetic encoding and mechanism of action of INK4A and ARF.

(Chudnovsky Y, et al, 2005)

RAF and RAS pathways

A genetic hallmark of melanoma is the presence of activating mutations in the oncogenes BRAF and NRAS, which are present in 70% and 15% of melanomas, respectively, and lead to constitutive activation of mitogen-activated protein kinase (MAPK) pathway signaling. However, molecules that inhibit MAPK pathway–associated kinases, like BRAF and MEK, have shown only limited efficacy in the treatment of metastatic melanoma. Thus, a deeper understanding of the cross talk between signaling networks and the complexity of melanoma progression should lead to more effective therapy.

NRAS mutations activate both effector pathways, Raf-MEK-ERK and PI3K-Akt in melanoma. The Raf-MEK-ERK pathway may also be activated via mutations in the BRAF gene. In a subset of melanomas, the ERK kinases have been shown to be constitutively active even in the absence of NRAS or BRAF mutations. The PI3K-Akt pathway may be activated through loss or mutation of the tumor suppressor gene PTEN, occurring in 30–50% of melanomas, or through gene amplification of the AKT3 isoform. Activation of ERK and/or Akt3 promotes the development of melanoma by various mechanisms, including stimulation of cell proliferation and enhanced resistance to apoptosis.


Figure 2: Schematic of the canonical Ras effector pathways Raf-MEK-ERK and PI3K-Akt in melanoma.

Curtin et al (2005) compared genome-wide alterations in the number of copies of DNA and mutational status of BRAF and NRAS in 126 melanomas from four groups in which the degree of exposure to ultraviolet light differs: 30 melanomas from skin with chronic sun-induced damage and 40 melanomas from skin without such damage; 36 melanomas from palms, soles, and subungual (acral) sites; and 20 mucosal melanomas. Significant differences were observed in number of copies of DNA and mutation frequencies in BRAF among the four groups of melanomas. Eighty-one percent of the melanomas on skin without sun-induced damaged had mutations in BRAF or NRAS. Melanomas with wild-type BRAF or NRAS frequently had increases in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1), downstream components of the RAS-BRAF pathway. Thus, the genetic alterations identified in melanomas at different sites and with different levels of sun exposure indicate that there are distinct genetic pathways in the development of melanoma and implicate CDK4 and CCND1 as independent oncogenes in melanomas without mutations in BRAF or NRAS. (Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47).

Genetic Heterogeneity of Melanoma

Melanoma exhibits molecular heterogeneity with markedly distinct biological and clinical behaviors. Lentigo maligna melanomas, for example, are indolent tumors that develop over decades on chronically sun-exposed area such as the face. Acral lentigenous melanoma, or the other hand, develops on sun-protected regions, tend to be more aggressive. Also, transcription profiling has provided distinct molecular subclasses of melanoma. It is also speculated that alterations at the DNA and RNA and the non-random nature of chromosomal aberrations may segregate melanoma tumors into subtypes with distinct clinical behaviors.

The melanoma gene atlas

Whole-genome screening technologies such as spectral karyotype analysis and array-CGH have identified many recurrent nonrandom chromosomal structural alterations, particularly in chromosomes 1, 6, 7, 9, 10, and 11 (Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47); however, in most cases, no known or validated targets have been linked to these alterations.

In A systematic high-resolution genomic analysis of melanocytic genomes, array-CGH profiles of 120 melanocytic lesions, including 32 melanoma cell lines, 10 benign melanocytic nevi, and 78 melanomas (primary and metastatic) by Chin et al (2006) revealed a level of genomic complexity not previously appreciated. In total, 435 distinct copy number aberrations (CNAs) were defined among the metastatic lesions, including 163 recurrent, high-amplitude events. These include all previously described large and focal events (e.g., 1q gain, 6p gain/6q loss, 7 gain, 9p loss, and 10 loss). Genomic complexity observed in primary and benign nevi melanoma is significantly less than that observed in metastatic melanoma (Figure 3)  (Chin L, et al, Genes Dev. 15 Aug 2006;20 (16):2149-2182).

Genetic heterogeneity Melanoma

Figure 3: Genome comparisons of melanocyte lesions (Chin L, et al, 2006)

Thus, genomic profiling of various melanoma progression types could reveal important information regarding genetic events those likely drive as metastasis and possibly, reveal provide cues regarding therapy targeted against melanoma.


  1. Greenlee RT, et al, Cancer J Clin. Jan-Feb 2001;51(1):15-36
  2. Weinstock MA, et al, Med Health R I. Jul 2001;84(7):234-6
  3. Chudnovsky Y, et al. J Clin Invest, 1 April 2005; 115(4): 813–824
  4. Jhappan C, et al, Oncogene, 19 May 2003;22(20):3099-112
  5. MacKie RM, et al, Lancet 26 Aug1989;2(8661):487-90)
  6. Gandini S, et al, Eur J Cancer, Sep 2005;41(14):2040-59)
  7. Curtin JA, et al, N Engl J Med, 17 Nov 2005;353(20):2135-47
  8. Chin L, et al, Genes Dev. 15 Aug 2006;20 (16):2149-2182

Related articles on Melanoma on this Open Access Online Scientific Journal, include the following: 

Thymosin alpha1 and melanoma Author/Editor- Tilda Barliya, Ph.D.

A New Therapy for Melanoma Reporter- Larry H Bernstein, M.D.

Melanoma: Molecule in Immune System Could Help Treat Dangerous Skin Cancer Reporter: Prabodh Kandala, Ph.D.

Why Braf inhibitors fail to treat melanoma. Reporter: Prabodh Kandala, Ph.D.

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Latest research efforts reported in the San Antonio Breast Cancer Symposium, 2012

Curator: Ritu Saxena, Ph.D.

‘Triple negative breast cancer’ or TNBC, as the name suggests, is a classification of breast cancers lacking the expression of estrogen receptor (ER) and progesterone receptor expression as well as amplification of the human epidermal growth factor receptor 2 (HER2).

Unlike other breast cancer types, treating TNBC is a challenge mainly because of the absence of well-defined molecular targets and because of disease heterogeneity. Currently, neoadjuvant chemotherapies are in use to treat TNBC patients. Some, around 30%, patients respond completely to neoadjuvant chemotherapy and have good outcomes after surgery. However, if there is a residual disease after therapy, outcomes are poor.

Therefore, current focus of the field is to first understand the complexity of the disease, both at genomic and molecular level and look for targets. Also, several combination chemotherapies are currently under trial to determine the efficacy, overall response rate, progression-free survival and other relevant factors for patients suffering with different forms of TNBC.

Recently, in the San Antonio Breast Cancer Symposium (SABCS 2012), several abstarcts related to TNBC research, both clinical and pre-clinical. Here is a compilation of some of the abstracts and their relevance in the field of TNBC research:

Triple Negative Breast Cancer: Subtypes, Molecular Targets, and Therapeutic Approaches, Pietenpol JA, Vanderbilt-Ingram Cancer Center; Vanderbilt University School of Medicine (Nashville, TN), Abstract no. ES2-2.

In order to better understand the complexity of TNBC, an integrative and comprehensive genomic and molecular analysis is required. The analysis would give important cues to developing and administering effective therapeutic agents. The group has compiled an extensive number of TNBC gene expression profiles and initiated molecular subtyping of the disease. Differential GE was used to designate 25 TNBC cell line models representative of the following subtypes:

  •  two basel-like TNBC subtypes with cell cycle and DDR gene expression signatures (BL1 and BL2);
  • two mesenchymal subtypes with high expression of genes involved in differentiation and growth factor pathways (M and MSL);
  • an immunomodulatory (IM) type;
  • a luminal subtype driven by androgen signaling (LAR)

The pharmacological drugs were chosen on the basis of the genetic pathways active in the cell lines with the abovementioned TNBC subtypes. It was observed that BL1 and BL2 subtype cell lines respond to cisplatin. Mesenchymal, basal, and luminal subtype lines with aberrations in PI3K signaling and have the greatest sensitivity to PI3K inhibitors.

The LAR subtype cell lines express AR and are uniquely sensitive to bicalutamide (AR antagonist). The experiment was a proof-of-concept that the best therapy could be based on TNBC subtypes.

The group has also developed a web-based subtyping tool referred to as “TNBCtype,” for candidate TNBC tumor samples using our gene expression metadata and classification methods. The approach would enable alignment of TNBC patients to appropriate targeted therapies.

The Clonal and Mutational Composition of Triple Negative Breast Cancers: Aparicio S, University of British Columbia (Vancouver, BC), Canada. Abstract no. ES2-3.

The abstract is on the same lines, TNBC heterogeneity that is. The concept of clonal heterogeneity in cancers, the spatial and temporal variation in clonal composition, is the focal point of the discussion. The group has developed next generation sequencing approaches and applied them to the understanding of mutational and clonal composition of primary TNBC. They have demonstrated that both mutational composition and clonal structure of primary TNBC is in fact a complete spectrum, a notion that is far from the previous one that stated TNBC to be a distinct disease. The authors add “clonal analysis suggests a means by which the genetic complexity might be reduced by following patient evolution over time and space.” The specific implications of the mutational and transcriptome landscapes of TNBC in relation to possible disease biologies were discussed in the symposium.

Profiling of triple-negative breast cancers after neoadjuvant chemotherapy identifies targetable molecular alterations in the treatment-refractory residual disease:

Balko JM, etal, Vanderbilt University (Nashville, TN); Foundation Medicine, (Cambridge, MA); Instituto Nacional de Enfermedades Neoplásicas, Lima, Peru

In the absence of hormone receptors and hence lack of targets, Neoadjuvant chemotherapy (NAC) is increasingly used in patients with TNBC. NAC can induce a pathologic complete response (pCR) in ∼30% of patients which portends a favorable prognosis. In contrast, patients with residual disease (RD) in the breast at surgical resection exhibit worse outcomes. The authors hypothesize that “profiling residual TNBC after NAC would identify molecularly targetable lesions in the chemotherapy resistant component of the tumor and that the persistent tumor cells would mirror micro-metastases which ultimately recur in such patients.” The researchers utilized targeted next generation sequencing (NGS) for 182 oncogenes and tumor suppressors in a CLIA certified lab (Foundation Medicine, Cambridge, MA) and gene expression profiling (NanoString) of the RD after NAC in 102 patients with TNBC. The RD was stained for Ki67, which has been reported to predict outcome after NAC in unselected breast cancers. Out of the 89 evaluable post-NAC tumors, 57 (64%) were basal-like; 19% HER2-enriched; 6% luminal A; 6% luminal B and 5% normal-like. Of 81 tumors evaluated by NGS, 89% demonstrated mutations in TP53, 27% were MCL1-amplified, and 21% were MYC-amplified.

Several pathways were found to be altered:

  • PI3K/mTOR pathway (AKT1-3, PIK3CA, PIK3R1, RAPTOR, PTEN, and TSC1)
  • Cell cycle genes (amplifications of CDK2, CDK4, and CDK6, CCND1-3, and CCNE1); loss of RB
  • DNA repair pathway (BRCA1/2, ATM)
  • Ras/MAPK pathway (KRAS, RAF1, NF1)
  • Sporadic growth factor receptor (amplifications occurred in EGFR, KIT, PDGFRA, PDGFRB, MET, FGFR1, FGFR2, and IGF1R.

NGS identified 7 patients with ERBB2 gene amplification. NGS could assist in the identification of ERBB2-overexpressing tumors misclassified at the time of diagnosis.

Amplifications of MYC were independently associated with poor recurrence-free survival (RFS) and overall survival (OS). In contrast to the earlier notion, high post-NAC Ki67 score did not predict poor RFS or OS in this predominantly TNBC cohort.

The authors concluded that “the diversity of lesions in residual TNBCs after NAC underscores the need for powerful and broad molecular approaches to identify actionable molecular alterations and, in turn, better inform personalized therapy of this aggressive disease.”

Identification of Novel Synthetic-Lethal Targets for MYC-Driven Triple-Negative Breast Cancer: Goga A, etal, UCSF (San Francisco, CA), Abstract No. S3-8

Reiterating the greatest challenge of the TNBC treatment, no targeted agents currently exist against TNBC. The group at UCSF has discovered that TNBC frequently express high levels of the MYC proto-oncogene. The discovery has led them to identify new “synthetic-lethal” strategies to selectively kill TNBC with MYC overexpression. “Synthetic lethality arises when a combination of mutations in two or more genes leads to cell death, whereas a mutation in only one of these genes has little effect. Using this strategy, we can take advantage of the elevated MYC signaling in TNBC to selectively kill them, while sparing normal tissues in which MYC is expressed at much lower levels”

The researchers performed a shRNA synthetic-lethal screen in the human mammary epithelial cells (HMEC), to identify new molecules, such as cell cycle kinases, which when inhibited can preferentially kill TNBC cells. A high-throughput screen of ∼2000 shRNAs, that target the human kinome (∼ 600 kinases) when performed, led to the identification of 13 kinases whose inhibition by >1 shRNAs gave rise to >50% inhibition of cell growth. ARK5 and GSK3A were the two other kinases that were shown to have a synthetic-lethal interaction with MYC. Since these two kinases have been identified in other studies, it gives validity to the ability to the methods of Goga etal in identifying synthetic-lethal targets. The group is currently characterizing and validating the 11 novel targets identified in this screen, using human cancer cell lines as well as mouse cancer models to determine the impact of inhibiting these targets on triple-negative breast cancer development and proliferation.


Dent R, etal.  Triple-negative breast cancer: clinical features and patterns of recurrence (2007) Clin Cancer Res 13, 4429-4434.

Lehmann BD, etal. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies (2011) J Clin Invest. 121: 2750-67.

Chen X, etal. TNBCtype: A Subtyping Tool for Triple- Negative Breast Cancer. (2012) Cancer informatics 11, 147-156.

Abstracts presented in SABCS 2012 can be accessed here.

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Author and Curator: Ritu Saxena, Ph.D.



Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived molecule that acts as a physiological messenger and has been known to regulate a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. Jordi Muntané et al

NO is synthesized by the Nitric Oxide synthase (NOS) enzyme and the enzyme is encoded in three different forms in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). The three isoforms, although similar in structure and catalytic function, differ in the way their activity and synthesis in controlled inside a cell. NOS-2, for example is induced in response to inflammatory stimuli, while NOS-1 and NOS-3 are constitutively expressed.

Regulation by Nitric oxide

NO is a versatile signaling molecule and the net effect of NO on gene regulation is variable and ranges from activation to inhibition of transcription.

The intracellular localization is relevant for the activity of NOS. Infact, NOSs are subject to specific targeting to subcellular compartments (plasma membrane, Golgi, cytosol, nucleus and mitochondria) and that this trafficking is crucial for NO production and specific post-translational modifications of target proteins.

Role of Nitric oxide in Cancer

One in four cases of cancer worldwide are a result of chronic inflammation. An inflammatory response causes high levels of activated macrophages. Macrophage activation, in turn, leads to the induction of iNOS gene that results in the generation of large amount of NO. The expression of iNOS induced by inflammatory stimuli coupled with the constitutive expression of nNOS and eNOS may contribute to increased cancer risk. NO can have varied roles in the tumor environment influencing DNA repair, cell cycle, and apoptosis. It can result in antagonistic actions including DNA damage and protection from cytotoxicity, inhibiting and stimulation cell proliferation, and being both anti-apoptotic and pro-apoptotic. Genotoxicity due to high levels of NO could be through direct modification of DNA (nitrosative deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage) and inhibition of DNA repair enzymes (such as alkyltransferase and DNA ligase) through direct or indirect mechanisms. The Multiple actions of NO are probably the result of its chemical (post-translational modifications) and biological heterogeneity (cellular production, consumption and responses). Post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation could lead to an increase in the cancer risk. This process can drive carcinogenesis by altering targets and pathways that are crucial for cancer progression much faster than would otherwise occur in healthy tissue.

NO can have several effects even within the tumor microenvironment where it could originate from several cell types including cancer cells, host cells, tumor endothelial cells. Tumor-derived NO could have several functional roles. It can affect cancer progression by augmenting cancer cell proliferation and invasiveness. Infact, it has been proposed that NO promotes tumor growth by regulating blood flow and maintaining the vasodilated tumor microenvironment. NO can stimulate angiogenesis and can also promote metastasis by increasing vascular permeability and upregulating matrix metalloproteinases (MMPs). MMPs have been associated with several functions including cell proliferation, migration, adhesion, differentiation, angiogenesis and so on. Recently, it was reported that metastatic tumor-released NO might impair the immune system, which enables them to escape the immunosurveillance mechanism of cells. Molecular regulation of tumour angiogenesis by nitric oxide.

S-nitrosylation and Cancer

The most prominent and recognized NO reaction with thiols groups of cysteine residues is called S-nitrosylation or S-nitrosation, which leads to the formation of more stable nitrosothiols. High concentrations of intracellular NO can result in high concentrations of S-nitrosylated proteins and dysregulated S-nitrosylation has been implicated in cancer. Oxidative and nitrosative stress is sensed and closely associated with transcriptional regulation of multiple target genes.

Following are a few proteins that are modified via NO and modification of these proteins, in turn, has been known to play direct or indirect roles in cancer.

NO mediated aberrant proteins in Cancer


Bcl-2 is an important anti-apoptotic protein. It works by inhibiting mitochondrial Cytochrome C that is released in response to apoptotic stimuli. In a variety of tumors, Bcl-2 has been shown to be upregulated, and it has additionally been implicated with cancer chemo-resistance through dysregulation of apoptosis. NO exposure causes S-nitrosylation at the two cysteine residues – Cys158 and Cys229 that prevents ubiquitin-proteasomal pathway mediated degradation of the protein. Once prevented from degradation, the protein attenuates its anti-apoptotic effects in cancer progression. The S-nitrosylation based modification of Bcl-2 has been observed to be relevant in drug treatment studies (for eg. Cisplatin). Thus, the impairment of S-nitrosylated Bcl-2 proteins might serve as an effective therapeutic target to decrease cancer-drug resistance.


p53 has been well documented as a tumor suppressor protein and acts as a major player in response to DNA damage and other genomic alterations within the cell. The activation of p53 can lead to cell cycle arrest and DNA repair, however, in case of irrepairable DNA damage, p53 can lead to apoptosis. Nuclear p53 accumulation has been related to NO-mediated anti-tumoral properties. High concentration of NO has been found to cause conformational changes in p53 resulting in biological dysfunction.. In RAW264.7, a murine macrophage cell line, NO donors induce p53 accumulation and apoptosis through JNK-1/2.


Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that is predominantly active under hypoxic conditions because the HIF-1a subunit is rapidly degraded in normoxic conditions by proteasomal degradation. It regulates the transciption of several genes including those involved in angiogenesis, cell cycle, cell metabolism, and apoptosis. Hypoxic conditions within the tumor can lead to overexpression of HIF-1a. Similar to hypoxia-mediated stress, nitrosative stress can stabilize HIF-1a. NO derivatives have also been shown to participate in hypoxia signaling. Resistance to radiotherapy has been traced back to NO-mediated HIF-1a in solid tumors in some cases.


Phosphatase and tensin homolog deleted on chromosome ten (PTEN), is again a tumor suppressor protein. It is a phosphatase and has been implicated in many human cancers. PTEN is a crucial negative regulator of PI3K/Akt signaling pathway. Over-activation of PI3K/Akt mediated signaling pathway is known to play a major role in tumorigenesis and angiogenesis. S-nitrosylation of PTEN, that could be a result of NO stress, inhibits PTEN. Inhibition of PTEN phosphatase activity, in turn, leads to promotion of angiogenesis.


C-src belongs to the Src family of protein tyrosine kinases and has been implicated in the promotion of cancer cell invasion and metastasis. It was demonstrated that S-nitrosylation of c-Src at cysteine 498 enhanced its kinase activity, thus, resulting in the enhancement of cancer cell invasion and metastasis.


Muntané J and la Mata MD. Nitric oxide and cancer. World J Hepatol. 2010 Sep 27;2(9):337-44.

Wang Z. Protein S-nitrosylation and cancer. Cancer Lett. 2012 Jul 28;320(2):123-9.

Ziche M and Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw. 2009 Dec;20(4):164-70.

Jaiswal M, et al. Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol Gastrointest Liver Physiol. 2001 Sep;281(3):G626-34.

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