COX-2 Inhibitor Slows Basal Cell Carcinoma

Image: Flickr - RBerteig
Image: Flickr - RBerteig
Researchers in the US have shown that the COX-2 inhibitor, celecoxib, can slow the development of basal cell carcinoma. Although celecoxib was known to inhibit the development of squamous cell carcinoma in mice, and epidemiological studies had suggested a protective role in people, little was known about its efficacy in preventing the more common basal cell carcinoma. In mice with a genetic mutation similar to that which occurs in Gorin syndrome – a genetic condition which predisposes to basal cell carcinoma – deletion of the COX-1 or COX-2 gene reduced the microscopic tumour burden by 75% when the mice were exposed to ionising radiation. Treatment with celecoxib reduced the tumour burden by 35%. The overall size of the tumours was also shown to be doubled in mice engineered to overexpress COX-2.

In a planned 3-year, double-blinded, randomised clinical trial in 60 patients with Gorin syndrome, a trend towards reduced basal cell carcinoma burden was seen in all subjects receiving oral celecoxib (200mg bid). If only patients with less severe disease (less than 15 lesions at study entry) were included in the analysis, celecoxib significantly reduced basal cell carcinoma number and burden: subjects receiving placebo had a 50% increase in burden per year whereas subjects in the celecoxib group had a 20% increase. The study began recruiting in 2001 and was discontinued in 2004 when rofecoxib was withdrawn from the market amidst concerns about an association between long-term treatment with COX-2 inhibitors and increased incidence of heart attack and stroke. At that time, most patients had received celecoxib for 2 years and none had suffered cardiovascular side effects as a result of participation in the trial. Although safety concerns appear to preclude oral dosing, the researchers hope that topical application of celecoxib could provide safer, yet still effective, protection against basal cell carcinoma.

The study is published in the January issue of Cancer Prevention Research.

Epidemiological studies have also linked COX-2 inhibitor use to reduced incidence of other cancers, including colorectal cancer and breast cancer. Inhibition of the COX-2 pathway has been shown to reduce cancer cell proliferation, increase apoptosis and reduce angiogenesis, as well as modifying the immune response.

Targeting Breast Cancer Stem Cells

Image: Flickr – BK1Bennett
Image: Flickr – BK1Bennett
Cancer stem cells (CSCs) have been identified in a variety of tumour types, including breast tumours, and have been proposed to be responsible for recurrence, resistance to chemotherapy and, perhaps, metastasis of cancers. Targeting of these CSCs in the treatment of cancer is therefore of great interest. The ability of the ionophore antibiotic, salinomycin, to kill breast tumour CSCs was recently reported and now collaborators from the University of Michigan Comprehensive Cancer Center and the Centre de Recherche en Cancérologie de Marseille have identified a new potential drug target.

In this latest study the team identified overexpression of CXCR1, the receptor for interleukin-8 (IL-8), by the CSC subpopulation in a breast cancer cell line. Furthermore, addition of recombinant IL-8 increased the CSC population and enhanced the propensity for invasion. Conversely, use of CXCR1-blocking antibodies or repertaxin, a small-molecule CXCR1 antagonist, selectively depleted the breast CSCs both in vitro and in murine xenograft models.

CXCR1 blockade also induced massive apoptosis in bulk tumour cells, mediated by FASL/FAS signalling. The effects on CSC viability as well as FASL production were mediated by the focal adhesion kinase/AKT/forkhead transcription factor FKHRL1 (FAK/AKT/FOXO3A) pathway. Importantly, administration of repertaxin reduced tumour growth and the development of systemic breast cancer metastasis in NOD/SCID mice.

The authors of the study, published in the Journal of Clinical Investigation, suggest blockade of CXCR1 as a novel target for depletion of CSCs, potentially enhancing the efficacy of chemotherapeutic regimes.

Spicing up Chemotherapy

Image: Flickr – Raveesh Vyas
We have previously reported on curcumin, the principal active ingredient of turmeric, which has been claimed to have activity against a variety of conditions. Now scientists at Wayne State University have explored the activity of curcumin against cancer stem cells (CSCs).

Colorectal cancer is the third most common form of cancer and, despite aggressive surgical intervention and chemotherapy, almost 50% of patients develop recurrent disease. In epithelial cancers, including colorectal cancer, growing evidence supports the hypothesis that tumour initiation and maintenance is driven by CSCs. Consequently, it is believed that failure to eliminate the underlying CSC population is responsible for cancer recurrence following therapy.

Curcumin (keto form)
Curcumin (keto form)
Curcumin has already been shown to have efficacy against a number of tumours in both animal models and early clinical trials. This latest study explored the activity of curcumin on colon cancer derived CSCs, either alone or in combination with the current standard treatment, FOLFOX (5-fluorouracil (5-FU) plus oxaliplatin). In cell culture experiments the researchers demonstrated that FOLFOX treatment increased the percentage of CSCs, presumably due to CSC-sparing. Treatment of these FOLFOX-surviving cells with curcumin alone or in combination with FOLFOX resulted in a marked reduction in the CSC population.

The study, published in Translational Oncology, went on to examine the potential mechanisms for the observed effects. The authors suggest that curcumin may represent a viable adjunct to current chemotherapy of colorectal cancer that could reduce rates of recurrence.

Hsp90 Inhibitor Effective in Models of non-Hodgkin’s Lymphoma

Histopathology of DLBCL occurring in the tonsil (CD20 immunostain) - Source: Wikimedia Commons
Histopathology of DLBCL occurring in the tonsil (CD20 immunostain) - Source: Wikimedia Commons
Around 85% of non-Hodgkin’s lymphomas in the United States are B-cell lymphomas and, of these, diffuse large B-cell lymphoma (DLBCL) account for about one in three cases. DLBCL is a fast growing lymphoma and only about half of people with this type of lymphoma are cured by current treatments, which include radiation therapy, chemotherapy and monoclonal antibodies.

PU-H71 structure
Researchers, including scientists from Weill Cornell Medical College, have now discovered that the heat shock protein inhibitor, PU-H71, selectively kills DLBCLs that depend on the B-cell lymphoma 6 protein (BCL-6) transcriptional repressor. BCL-6 is involved in the pathogenesis of around 70% of cases of DLBCL. BCL-6 and heat shock protein (Hsp90) were almost invariantly co-expressed in the nuclei of primary DLBCL cells and a complex of Hsp90 and BCL-6 was found to stabilise BCL-6 mRNA and protein. Hsp90 inhibitors allowed transcription of genes normally repressed by BCL-6 and a stable mutant of BCL-6 was found to rescue DLBCL cells from Hsp90 inhibitor–induced apoptosis. In mouse xenograft models, PU-H71 was shown to preferentially accumulate in lymphomas compared to normal tissues, and led to almost complete tumour regression by allowing reactivation of key BCL-6 target genes and inducing apoptosis. PU-H71 shows very low toxicity in animal models and the researchers hope that the compound will be similarly well tolerated and efficacious in human patients.

The study is published in Nature Medicine.

PU-H71 has previously been shown to induce complete responses in triple-negative breast cancer models. Triple-negative breast cancers are defined by a lack of expression of estrogen, progesterone or HER2 receptors and are currently treated with conventional chemotherapy which is effective only in some patients, leaving others with high rates of early relapse.

Mapping Phosphorylation in Cancer

Phosphorylation is a key mechanism for regulation of protein activity and the phosphorylation of tyrosine residues, in particular, is important in signalling pathways. Aberrant phosphorylation has been observed in many cancers and has driven the development of kinase inhibitors that have utility in a number of cancer subtypes. Since abnormal activation of signalling pathways is a common feature that accompanies tumour initiation and progression, methods to assess signalling pathway status of cancer tissue relative to normal could provide important insights.

A group of US researchers has now conducted a detailed analysis of tyrosine phosphorylation states in lung cancer compared to normal tissue. The work documents a large set of sites that are differentially phosphorylated in the cancer tissue, immediately providing a number of drug target candidates. Taking the study a step further, the group has developed computational methodology to identify signalling pathways where phosphorylation activity is strongly correlated with the lung cancer phenotype.

The EGFR signalling pathway. Pink indicates higher phosphorylation in tumour samples, while green indicates higher phosphorylation in normal tissue samples. Full details can be found in PLoSone.
The EGFR signalling pathway. Pink indicates higher phosphorylation in tumour samples, while green indicates higher phosphorylation in normal tissue samples. Full details can be found in PLoSone.

The results provide a highly predictive set of signatures that reliably distinguish each lung cancer from normal. Since the signatures identify proteins and pathways where phosphorylation should be inhibited, the methodology highlights potential new targets for drug discovery.

The study is published in the journal PLoSone.

Engineering New Cancer Treatments

Image: Flickr - royblumenthal
Image: Flickr - royblumenthal
Metastasis, the spread of cancer cells to new organs, generally signals a poor prognosis but detecting circulating tumour cells is both difficult and time consuming. US and Russian scientists have now described a way to magnetically capture circulating tumour cells in the bloodstream that has potential for the early diagnosis of cancer and the prevention of metastasis. Magnetic nanoparticles, which were functionalised to target a receptor commonly found on breast cancer cells, were shown to bind and capture circulating tumour cells in the bloodstream when injected into mice. To improve detection sensitivity and specificity, gold-plated carbon nanotubes conjugated with folic acid were used as a second contrast agent for photoacoustic imaging. The approach allows cancer cells from a large volume of blood to be concentrated in peripheral blood vessels by a magnet attached to the skin, potentially increasing specificity and sensitivity up to 1,000-fold compared to existing technology. The cells can then be removed by microsurgery for genetic analysis, or can be noninvasively eradicated directly in blood vessels by laser irradiation through the skin, which is safe for normal blood cells.

The study is published in Nature Nanotechnology.

In a separate study, published in the British Journal of Cancer, a non-pathogenic strain of Salmonella typhimurium has been used to deliver the cytotoxic protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) directly to solid tumours. S. typhimurium were engineered to secrete murine TRAIL under control of the radiation-inducible RecA promoter which activates when cells experience DNA damage. Common bacteria such as Salmonella and Escherichia favour the microenvironment of solid tumours over normal tissue and when the modified S. typhimurium were injected into mice with mammary tumours they localized to the tumour. After 48 hours, the bacteria had multiplied to about 10 million per tumour and the mice were exposed to a very low dose of γ-radiation. The resulting mild DNA damage (single-stranded breaks) activated RecA and initiated synthesis of TRAIL which is highly toxic to cancer cells. Mice that received two low dose radiation treatments had the best result since TRAIL clears quickly and its release must be regularly re-stimulated for best effect; repeated dosing with modified S. typhimurium in conjunction with low dose radiation improved the 30-day survival from 0 to 100%.

The researchers hope that, once the technique is fully developed, spatial and temporal control of the release of cytotoxic agents will provide enhanced efficacy while limiting toxicity.

Targeting ‘Normal’ Cells to Treat Cancer

Crystal structure of human FAP - pdb ID=1z68
Crystal structure of human FAP - pdb ID=1z68
Most cancer treatments directly target the cancerous cells but tumours are a heterogeneous mix of transformed cells and ‘normal’ cells. Stromal cells, including infiltrating inflammatory and immune cells, endothelial cells, pericytes, and tumour-associated fibroblasts (TAFs), play a key role in cancer growth and progression, both directly though cell contact and indirectly through paracrine/exocrine signalling, protease activity and modulation of the extracellular matrix. Identifying stromal targets could provide a complementary strategy to directly targeting cancer cells but the widespread expression of many proteases limits their potential for therapeutic intervention.

A team of scientists led by researchers at the Wistar Institute has now identified a critical role for fibroblast activation protein (FAP), expressed by TAFs, in promoting tumour growth in mice. FAP is a type II cell surface serine protease expressed predominantly by fibroblastic cells in areas of active tissue remodelling such as tumour stroma, fibrosis, or healing wounds. FAP is a member of the post-proline dipeptidyl aminopeptidase family, sharing the highest similarity with dipeptidyl peptidase IV (DPPIV) but, unlike DPPIV, FAP is not found in benign lesions or normal adult tissues.

PT630 structure
The team found that the enzymatic activity of endogenous FAP promoted tumour growth in mouse models of lung and colon cancer and, moreover, that genetic deletion of FAP expression, or pharmacological inhibition of FAP activity using PT630, was able to significantly inhibit tumour cell proliferation. Inhibition of FAP activity was further shown to disrupt the organisation of collagen fibres in the extracellular matrix and decrease blood vessel density in tumours.

FAP is expressed in 90% of solid tumours and the study, which is published in the Journal of Clinical Investigation, suggests that therapies that target stromal cells could increase the effectiveness of cancer cell-targeted therapies for solid tumours.

Antibody Recruiting Molecules Target HIV, Cancer

hiv virus cartoon Both HIV and cancer cells have found ways to evade the body’s immune system but researchers at Yale University have now found a way to boost the body’s ability to fight HIV and cancer. The team have identified bifunctional small molecules, termed “antibody-recruiting molecule targeting HIV” (ARM-H) and “antibody-recruiting molecule targeting prostate cancer” (ARM-P), which bind simultaneously to antibodies and to proteins on HIV, HIV-infected cells or cancer cells.

ARM-H molecules bind to gp120, a component of the Env glycoprotein on the surface of HIV and virus-infected cells and to anti-2,4-dinitrophenyl antibodies already present in the bloodstream. The ternary complex formed between the antibody, ARM-H, and gp120 is immunologically active, and leads to complement-mediated destruction of Env-expressing cells. ARM-H also prevents virus entry into human T-cells and so has the potential to inhibit viral replication by two mutually reinforcing mechanisms.

ARM-P molecules bind with high affinity to prostate-specific membrane antigen (PSMA) and, by inducing complexes of anti-2,4-dinitrophenyl antibodies with prostate cancer cells, mediate antibody-dependent killing of the cancer cells.

The team has begun to evaluate the ARM molecules in mice, and hope that the strategy of using antibody-recruiting small molecules to boost the immune response will prove useful for treating HIV, cancer, and other diseases.

Both studies are published in the Journal of the American Chemical Society (ARM-H and ARM-P).

Suppressing ROCK May Prevent Breast Cancer Metastases

Image: Flickr - Austin UK
Image: Flickr - Austin UK
Nearly half of all patients treated for apparently localised breast cancer develop metastatic disease. Although there are treatment options that prolong survival and improve quality of life for patients with metastatic breast cancer, these treatments rarely lead to long-term survival without disease recurrence. The most common sites of metastases are the bones, the liver and the lungs: breast cancer is the most common origin of metastatic deposits in the skeleton.

Researchers from Tufts University have now identified Rho-associated kinase (ROCK) as a potential target to reduce breast cancer metastasis. Signalling through ROCK plays a key role in regulating cell adhesion and motility and aberrant expression of ROCK has been linked to metastasis. In the present study, the team showed that ROCK expression is increased in metastatic human mammary tumours and breast cancer cell lines compared with non-metastatic tumours and cell lines. A metastatic phenotype could also be induced in a cell line that is not normally metastatic by over-expression of ROCK.

In a novel mouse model of “human breast cancer metastasis to human bone”, inhibiting ROCK in the earliest stages of breast cancer decreased metastatic tumour mass in bone by 77% and overall frequency of metastasis by 36%. ROCK function could be effectively blocked by either ROCK-targeting short hairpin RNA (shRNA) or by the specific ROCK inhibitor, Y27632. Expression of the microRNA cluster, c-Myc-regulated miR-17-92 was found to be elevated in metastatic breast cancer cells compared with non-metastatic cells and reduced by treatment with Y27632. Blockade of miR-17 was further shown to decrease breast cancer cell invasion/migration in vitro and metastasis in vivo. The authors suggest that the effects of ROCK may be mediated by modulating the c-Myc pathway, including c-Myc-dependent microRNAs. They propose that inhibition of ROCK, or the pathway it stimulates, may represent a novel approach for treatment of breast cancer metastases.

The study is published in the journal Cancer Research.

Syk Protective Against Breast Cancer

Inhibitors of the non-receptor spleen tyrosine kinase (Syk) are being developed to treat a variety of allergic and autoimmune disorders, as well as some types of cancer, but researchers at Georgetown University Medical Center have cautioned that Syk controls the growth of normal breast cells and prevents the development of breast cancer.

Although it is not known what causes loss of Syk function, Syk is negatively correlated with invasion and metastasis of tumour cells and, as breast tumours progress, more and more Syk protein is lost. Since total knock-out of Syk is perinatally lethal, the researchers created mice with only one copy of the gene and found that loss of the single allele led to increased proliferation and invasion of normal breast cells in the mouse mammary gland during puberty, and resulted in development of breast cancer in adulthood.

Three colour confocal images of cells cultured on crosslinked gelatin showing the distribution of DAPI (nuclei, blue), phalloidin (F-act, green), and vimentin (magenta) in control (left) versus Syk siRNA transfected MCF10A cells (right). Scale bars = 20 µm. Image: PLoSone
Three colour confocal images of cells cultured on crosslinked gelatin showing the distribution of DAPI (nuclei, blue), phalloidin (F-act, green), and vimentin (magenta) in control (left) versus Syk siRNA transfected MCF10A cells (right). Scale bars = 20 µm.

Image: PLoSone

siRNA or shRNA knockdown of Syk protein in cultures of normal human breast epithelial cells also dramatically increased proliferation and invasion. Syk loss was shown to release inhibition of a number of signalling pathways that are normally repressed in epithelial cells and that promote increased proliferation, motility, and invasiveness. The findings, which were published on October 15th in the journal PLoS ONE, suggest that Syk plays an important role in controlling growth as breast tissue develops and acts as a tumour suppressor for breast cancer. The finding that only partial loss of Syk function in mice was sufficient to induce mammary carcinomas underscores the potential risk of Syk inhibition in promoting breast cancer. The team hope that identification of the pathways that are negatively regulated by Syk will ultimately provide new targets for the treatment of breast cancer.