oncology – Page 5 – Drug Discovery Opinion

New Target for Malignant Melanoma

Sun photo by Radiant Guy — Photo: Radiant Guy

Malignant melanoma is the most aggressive form of skin cancer, involving malignant transformation of melanocytes. Although it is one of the less common types of skin cancer, it accounts for around 75% of skin cancer-related deaths. The greatest chance of survival currently depends on early diagnosis and surgical removal of the tumour.

Melanocyte differentiation is under the control of microphthalmia-associated transcription factor (MITF), a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. Paradoxically, however, high expression of MITF also has an anti-proliferative effect. Scientists at Keio University School of Medicine, Shinjuku-ku, Tokyo and collaborators have demonstrated that both depletion and forced expression of MITF in human melanoma cell lines significantly inhibited proliferation. Although approximately half of the cell lines were resistant to MITF depletion, simultaneous depletion of both MITF and BRAF^V600E, an oncogenic kinase also associated with melanoma, significantly inhibited melanoma growth even in the resistant cell lines. The work, published in the October edition of Cancer Science, suggests that dual inhibition of MITF and BRAF^V600E represents a useful strategy for treatment of melanoma.

A second study, from collaborators at The Scripps Research Institute and the Genomics Institute of the Novartis Research Foundation, has found that the receptor tyrosine kinase, TYRO3, is an upstream regulator of MITF expression. TYRO3 expression is significantly elevated in human primary melanoma tissue samples and melanoma cell lines and correlates with MITF mRNA levels. Their study showed that TYRO3 overexpression bypassed BRAF^V600E-induced senescence in primary melanocytes, inducing transformation of non-tumourigenic cell lines. Knockdown of TYRO3 repressed cellular proliferation of melanoma cells and sensitised them to chemotherapy-induced apoptosis. In addition, TYRO3 knockdown in melanoma cells also inhibited tumourigenesis in vivo. The study is published in the 23^rd September online edition of PNAS.

Taken together, the two studies suggest that a dual TYRO3/ BRAF^V600E inhibitor would be interesting to evaluate in melanoma.

Sugar Coating Works for Nanoparticles

Raising the temperature of human tissue by as little as 5^oC damages or kills cells, and thermal ablation offers an alternative to chemotherapy or radiotherapy for the treatment of cancer. The main challenge in the clinical application of thermal therapy is how to selectively cause necrosis within a tumour, without overheating and damaging surrounding tissue. Magnetic nanoparticles, particularly if they can be targeted selectively to a tumour, offer the potential to deliver heat in a site-selective, and even cell-selective, manner. Nanoparticles for biomedical applications are most often formed using magnetite (Fe₃O₄) and their manufacture requires the synthesis of stable colloidal suspensions in biocompatible fluids (water or saline) that maintain their stability in blood or plasma. The particles must also produce a predictable and sufficient amount of heat when modest particle concentrations are exposed to alternating magnetic fields at amplitudes that can be applied safely to large areas of tissue. These criteria mean that the surface chemistry, size, and magnetic properties of the particles must be tightly controlled during manufacture.

Many scientists had believed that the best results would be achieved with small, non-interacting particles which can evade the body’s immune system, but a study led by researchers at the National Institute of Standards and Technology (NIST) has shown that dextran-coated magnetic particles that have good in vivo efficacy interact strongly with each other. In a mouse breast cancer model, three out of four mice treated with a single dose of the particles displayed a complete response, with no tumour re-growth during the 60 day study period. To produce the particles, a dextran shell was physically adsorbed onto an iron oxide core, giving particles with a diameter (core + shell) of ca 100nM. Extensive characterisation of the particles revealed a preferred interparticle spacing, consistent with chain-forming interactions between the particles. Although the magnetic cores of the particles attract each other, fibres from the dextran coat prevent the particles from clumping together and eliciting an immune response. When ingested by tumour cells, the association between the particles is close enough to increase their heat-generating efficiency when subjected to an alternating magnetic field.

An iron-centered nanoparticle (left) analyzed at NISTs Center for Neutron Research has a coating of the sugar dextran, whose tendrils prevent groups of the particles from clumping. When tumour cells ingest them (right), the particles still congregate closely enough to share heat when stimulated by a magnetic field, killing the cells. White arrow indicates a red blood cell. Credit: NIST — An iron-centered nanoparticle (left) analyzed at NIST's Center for Neutron Research has a coating of the sugar dextran, whose tendrils prevent groups of the particles from clumping. When tumour cells ingest them (right), the particles still congregate closely enough to share heat when stimulated by a magnetic field, killing the cells. White arrow indicates a red blood cell. Credit: NIST

The findings, which are published in the journal Nanotechnology, may help in the design of better nanoparticles for the treatment of cancer.

PARP Inhibitors – More Widely Effective than First Thought

The PARP (poly (ADP-ribose) polymerase) family of nuclear enzymes play key roles in a variety of cellular processes, including DNA repair. Although DNA repair is beneficial in normal, non-cancerous cells, PARP expression and activity are significantly up-regulated in many cancers, suggesting that cancer cells may rely more than normal cells on the activity of PARP, likely because of defects in other DNA repair pathways. PARP inhibitors, including BSI-201 (BiPar Sciences) and olaparib (AstraZeneca) are already showing promise in the treatment of breast, ovarian and prostate cancers linked to faulty BRCA1 and BRCA2 proteins which normally act as tumour suppressors by repairing damaged DNA.

olaparib and bsi-201 structures In a study published online on September 16^th in the journal EMBO Molecular Medicine, scientists from the Breakthrough Breast Cancer Research Centre at the ICR and the Lombardi Comprehensive Cancer Center, Washington have now shown that PARP inhibitors can also kill cancer cells with mutations in the PTEN (phosphatase and tensin homolog) gene. As with BRCA1/2, the protein encoded by the PTEN gene functions as a tumour suppressor. Cells with faulty PTEN genes were found to be up to 25 times more sensitive to PARP inhibitors than normal cells.

PTEN deficiency was found to cause a homologous recombination (HR) defect in human tumour cells. Amongst other roles, HR acts as a natural safety mechanism that protects chromosomes against damage to the two DNA strands, such as double-strand breaks. Dysfunctions of proteins that facilitate HR are strongly associated with increased susceptibility to several types of cancer – mutations in BRCA1 and BRCA2 also lead to deficiencies in HR. The HR deficiencies caused by reduced PTEN activity were shown to sensitise tumour cells to the effects of PARP inhibitors, both in cell culture experiments and in mouse studies. PTEN is one of the most commonly mutated genes in human cancers and, if clinical trials show that PARP inhibitors are effective in treating patients with PTEN-related tumours, this approach could potentially treat a greater variety of cancers than had previously been thought.

Retrovirus Linked to Prostate Cancer

A number of human cancers are linked to viral infection including cervical cancer (human papilloma virus), liver cancer (hepatitis B and C viruses), and lymphoma (Epstein Barr virus). The human retrovirus, XMRV (xenotropic murine leukemia virus-related virus) has previously been found in prostate cancers – most often in men with a defect in the antiviral defence protein, ribonuclease L – but, since only malignant tissues were analysed, a direct association with prostate cancer was not established. In a study published in the September 7^th online edition of PNAS, researchers at Columbia University and the University of Utah have now analysed specimens from 233 cases of prostate cancer for the presence of XMRV and compared them with 101 benign control specimens. XMRV infection was found to be associated with prostate cancer, especially with more aggressive tumours, and infection was also found to be independent of reduced activity of ribonuclease L. The virus was detected in 27% of prostate cancer specimens compared with 6% of benign tissue samples and was found almost exclusively in malignant cells.

XMRV is a member of the gammaretrovirus family, other members of which are known to cause cancers in rodents, cats and primates. The study suggests that XMRV infection could contribute to the development of human prostate cancer, although the mechanism by which the virus transforms cells remains to be fully elucidated. As with other retroviruses, XMRV integrates into host DNA and it may be that it inserts close to a gene that regulates cell growth. XMRV was found to replicate efficiently in a cell line derived from human prostate cancer but not in other human cell lines, suggesting a viral tropism that needs to be investigated further. If further studies provide a causal link between XMRV and the development of prostate cancer, opportunities for diagnostic tests, antiretroviral therapy, or ultimately a vaccine against prostate cancer, could follow.

Two Faces of FOXO3a in Cancer

FOXO3a is a member of the O subclass of fork-head transcription factors, so-called because of their distinctive forked DNA binding domain. It is negatively regulated by phosphorylation through the PI3K/Akt pathway, which confines the transcription factor to the cytoplasm. Generally, FOXO3a is considered to be pro-apoptotic and, as such, a tumour suppressor. The PI3K/Akt pathway is up-regulated in many cancers, consistent with reduced transcriptional activity of FOXO3a. Further, FOXO3a has been reported to mediate the cytotoxic activity of chemotherapeutic agents such as cisplatin and paclitaxel.

In contrast, a pro-survival role for FOXO3a in oxygen-deprived cells has also been reported (Molecular Cell 28, 941–953, December 28, 2007). In this study, from scientists at the Campbell Family Institute for Breast Cancer Research, University Health Network, Ontario Cancer Institute and Princess Margaret Hospital, Toronto, FOXO3a inhibited HIF-1 induced apoptosis in both normal and cancer cells under hypoxic conditions.

Now new research from the Mayo Clinic in Florida and Harvard Medical School has shown that FOXO3a promotes invasive migration of tumour cells in response to hypoxia, mediated by induced expression of matrix metalloproteinases (Molecular and Cellular Biology, September 2009, p. 4906-4917, Vol. 29, No. 18). This tumour cell migration ultimately opens the door to metastasis.

Clearly a pro-apoptotic or pro-survival role for FOXO3a is dependent on context. The challenge for drug discovery is to determine if, when and how therapeutic intervention in this pathway is viable.

Angiotensin 1-7 Inhibits Lung Cancer Tumour Growth in Mice

The renin-angiotensin system was originally believed to be a relatively straightforward cascade involved primarily in controlling blood pressure and was exploited to provide a number of antihypertensive medicines. Angiotensinogen is cleaved by renin to give angiotensin I (Ang I), followed by further cleavage of Ang I by angiotensin-converting enzyme 1 (ACE) to give the octapeptide angiotensin II (Ang II) which binds to angiotensin receptor 1 (AT₁R) or 2 (AT₂R). AT₁R mediates most of the cardiovascular effects of Ang I and activation leads to increased blood pressure which can be countered by the action of renin inhibitors, ACE inhibitors or AT₁R antagonists.

More recently, a parallel pathway in which Ang I is cleaved by angiotensin-converting enzyme 2 (ACE2) to give the heptapeptide, angiotensin 1-7 [Ang-(1-7)], has been discovered. This second pathway, in which Ang-(1-7) binds to the Mas receptor, is believed to act as a counter-regulatory axis to the ACE 1 – Ang II – AT₁R axis. Roles have also been proposed for Ang-(1-7) and Mas in kidney disease, liver disease, and spermatogenesis.

Ang-(1-7) is also known to have anti-proliferative properties and to inhibit angiogenesis and researchers at Wake Forest University School of Medicine have now shown that Ang-(1-7) reduces lung tumour growth and inhibits blood vessel formation in mice. The team had previously shown that Ang-(1-7) inhibits the growth of human lung cancer cells in vitro and reduces the size of human lung tumour xenografts in vivo. In the present study, daily subcutaneous administration of Ang-(1-7) for six weeks was found to significantly reduce tumour growth by human lung carcinoma A549 cells and also markedly decrease vessel density compared with saline treated controls. The study, which is published in the August issue of Molecular Cancer Therapeutics, suggests that Ang-(1-7) may provide a novel and effective treatment for lung cancer. The team have also shown that Ang-(1-7) is effective against breast, colon and brain tumours. A clinical trial of Ang-(1-7) has been completed at the School of Medicine and the results are currently being reviewed.

Intestinal Flora may Trigger Colon Cancer

Just as Helicobacter pylori, which causes stomach ulcers and gastric cancers, is being eradicated from many developed countries, Johns Hopkins scientists have shown that bacteria that cause diarrhoea may also lead to some colon cancers. Enterotoxigenic strains of Bacteroides fragilis (ETBF) asymptomatically colonise a proportion of the human population but can also cause inflammatory diarrhoea in both children and adults. An earlier study in Turkey had linked ETBF infection to colon cancer and, to further understand this association, the Johns Hopkins team have carried out a study in multiple intestinal neoplasia (Min) mice. These animals carry mutations in the APC (adenomatosis polyposis coli) gene and spontaneously develop multiple small intestinal adenomas as well as more sporadic colonic adenomas. Mutations in the tumour-suppressing APC gene are also associated with human colon cancer. The present study showed that, although both ETBF and nontoxigenic B. fragilis (NTBF) chronically colonise mice, only ETBF causes diarrhoea and inflammation and induces colonic tumours. The diarrhoea resolved quickly but the mice developed colitis within 7 days and, after 4 weeks, had numerous colonic tumours. ETBF was found to strongly activate Stat3 in the colon, leading to a dramatic (100-fold higher than normal) and selective T_H17 response. Blocking IL-17 as well as the receptor for IL-23, a key cytokine amplifying T_H17 responses, inhibited the colitis, colonic hyperplasia and tumour formation triggered by ETBF. The study, which is published in the August 23^rd issue of Nature Medicine, provides new mechanistic insights into the development of human colon cancers and may lead to the development of vaccines or improved therapies.

Salinomycin Selectively Attacks Breast Cancer Stem Cells

There is growing evidence for a subpopulation of cells in breast cancer, known as breast cancer stem cells, that may be responsible for disease recurrence, resistance to conventional treatments, and perhaps metastasis. A team of US scientists has now shown that it is possible to selectively kill breast cancer stem cells (CSCs) using the ionophore antibiotic, salinomycin.

Patient-derived CSCs are difficult to collect and maintain in culture and a key aspect of the work was an ability to generate relatively homogenous and stable populations of CSC-like cells that could then be used for screening. Induction of epithelial-mesenchymal transition (EMT) in normal or neoplastic mammary epithelial cell populations had previously been shown to result in the enrichment of cells with stem cell-like properties and the researchers showed that normal and cancer cell populations experimentally induced into EMT also show increased resistance to chemotherapy drug treatment. Using two populations of mammary epithelial cells – one that had been induced to undergo EMT and one which had not – the team screened around 16,000 compounds and found that just 32 of them showed selective toxicity towards the cells that had undergone EMT. On the basis of potency and selectivity, salinomycin was chosen for further studies and was also found to decrease the proportion of CSCs that occur naturally as a subpopulation of breast cancer cells. Pre-treatment with salinomycin was found to decrease the tumour-seeding ability of cancer cell lines more than 100-fold compared with paclitaxel pre-treatment, and salinomycin also reduced mammary tumour size in mice to a greater extent than the chemotherapy drug, paclitaxel, or vehicle. Similar methodology could potentially be used to identify compounds that selectively kill CSCs from other types of cancer including leukemia, brain cancer, lung cancer, prostate cancer and melanoma, but it will be important to extend the findings to primary tumour cells from patients.

Salinomycin is used in farming to prevent coccidiodomycosis in poultry, and to alter gut flora in order to improve nutrient absorption in ruminants. The compound interferes with potassium transport across mitochondrial membranes, reducing intracellular energy production. It may also disrupt Na⁺/Ca²⁺ exchange in skeletal, and in some cases, cardiac muscle, allowing a fatal accumulation of intracellular calcium. Cases of animal poisoning with ionophore antibiotics have been widely described and the symptoms, which include progressive muscle weakening, appear to be similar to those seen following accidental exposure in man. Although the current study focussed largely on salinomycin, around 30% of the primary screen hits were confirmed on retesting, and expanding the screen may provide active compounds that may be more suitable for further development. The mechanism(s) by which salinomycin exhibits selective toxicity for CSCs is not known, but less toxic compounds that selectively target CSCs – but not normal stem cells – could find a place in future cancer therapies.

The study was published in the August 13^th advance online issue of Cell.

Mitochondrial Dysfunction and Polo-Like Kinases

The observation that cancer cells have a high rate of glycolysis, even in the presence of oxygen, was first made by Otto Warburg in 1924. Warburg assumed that the reason for this was a mitochondrial malfunction in cancer cells that drove a dependency on anaerobic glycolysis to generate ATP. Subsequently, it has been shown that the Warburg Effect is in operation even in cancer cells with intact mitochondria. It has been hypothesised that cancer cells exploit the less efficient glycolytic route to ATP to meet the high energy requirements of rapid proliferation and/or to provide a defence against the highly oxidative environment in which they survive. Indeed, increased glucose metabolism provides cancer cells with high levels of antioxidants, ATP and metabolites for growth. Down-regulation of mitochondrial activity further protects cancer cells from apoptosis. Understanding of the mechanisms underlying the Warburg Effect is still far from complete (recently reviewed), but the pathways remain an intense area of study to identify new approaches to cancer chemotherapy.

Scientists at the Translational Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health and Kyoto Prefectural University of Medicine, Kyoto have now reported that ablation of mitochondrial respiration markedly increases expression of Polo-like kinase 2 (PLK2). In these cells, PLK2 is required for in vitro growth and the PLK2-mediated phosphorylation of Ser-137 of Polo-like kinase 1 (PLK1) is sufficient for survival. The researchers also showed that, in vivo, knockdown of PLK2 in an isogenic human cell line with a modest defect in mitochondrial respiration eliminated xenograft formation, indicating that PLK2 activity is necessary for growth of cells with compromised respiration.

The study is reported in the August 11^th early edition of Proceedings of the National Academy of Sciences.

Metformin Linked to Reduced Cancer Risk

Pancreatic cancer has one of the highest fatality rates of all cancers and is the fourth leading cause of cancer-related deaths in the United States; more than 35,000 people were estimated to have died from the disease in 2008. Patients diagnosed with pancreatic cancer typically have poor prognoses because the cancer is difficult to detect in its early stages – there are few symptoms and any that do present are vague and often go unnoticed. Although the exact causes of pancreatic cancer are not understood, the disease is more common in people with type 2 diabetes, although the reasons for this link are also unclear.

A recent study by researchers at the University of Texas M.D. Anderson Cancer Center has shown, however, that diabetics who have taken the anti-diabetic drug, metformin, alone or in combination with other drugs, have a 60% lower risk of developing pancreatic cancer compared with patients who have never taken it. Metformin is the most popular anti-diabetic drug in the United States and works primarily by suppressing glucose production by the liver and by increasing insulin sensitivity.

The study, which is published in the journal Gastroenterology, also suggested some increased risk of developing pancreatic cancer in patients who received insulin or insulin secretagogues, such as sulfonylureas and meglitinides. The study population was too small to determine the effects of another class of anti-diabetic drugs, the thiazolidinones.

The study evaluated prior use of antidiabetic drugs in just over 1800 people, 973 with pancreatic adenocarcinoma (including 259 diabetic patients) and 863 controls (including 109 diabetic patients). Use of metformin was found to be associated with a significantly decreased risk of pancreatic cancer compared with metformin non-use (adjusted odds ratio 0.38) whereas long-term insulin use was associated with a moderately higher risk of pancreatic cancer compared to insulin non-use (adjusted odds ratio 2.78). The association between pancreatic cancer and long term insulin use was, however, based on only 17 cases and 9 controls and needs to be investigated further in a larger study. The impact of insulin secretagogues on risk of pancreatic cancer likewise needs to be fully assessed in a larger study. Although it was not possible to gauge the effect of severity of diabetes or effectiveness of diabetes control on the results, the study does, however, show a robust protective effect of metformin, especially after long term use, against pancreatic cancer in people with type 2 diabetes.