Rena G. Lapidus

The Loss of Estrogen and Progesterone Receptor Gene Expression in Human Breast Cancer

Hormone responsiveness is a critical determinantof breast cancer progression and management, and theresponse to endocrine therapy is highly correlated withthe estrogen receptor (ER)3 and progesterone receptor (PR) status of tumor cells. Thus, keyareas of study in breast cancer are those mechanismsthat regulate ER and PR expression in normal andmalignant breast tissues. One-third of all breastcancers lack ER and PR; these conditions are associatedwith less differentiated tumors and poorer clinicaloutcome. In addition, approximately one-half ofER-positive tumors lack PR protein and patients withthis phenotype are less likely to respond tohormonal therapies than those whose tumors express bothreceptors. Since PR is induced by ER; its presence is amarker of a functional ER. In this review, we will discuss possible mechanisms for loss of ER andPR gene expression, especially structural changes withineach gene including deletions, polymorphisms ormethylation. Improved understanding of the pathways that lead to loss of ER and/or PR proteinsshould allow the development of better predictiveindicators as well as novel therapeutic approaches totarget these hormone-independent cancers.

Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma

Poly(ADP‐ribose) polymerase (PARP) inhibitors enhance the antitumor activity of the topoisom‐erase I inhibitor irinotecan (CPT‐11), which is used to treat advanced colorectal carcinoma. Since PARP inhibitors sensitize tumor cells also to the methylating agent temozolomide (TMZ) and clinical trials are evaluating CPT‐11 in combination with TMZ, we tested whether the PARP inhibitor GPI 15427 (10‐(4‐methyl‐piperazin‐1‐ylm‐ethyl)‐2H‐7‐oxa‐1,2‐diaza‐benzo[de]anthracen‐3‐one) increases the efficacy of CPT‐11 + TMZ against colon cancer. Moreover, due to the ability of PARP inhibitors to avoid cell death consequent to PARP‐1 overactivation, we evaluated whether oral administration of GPI 15427 provides protection from the dose‐limiting intestinal toxicity of CPT‐11. The results of colony formation assay indicated that GPI 15427 increased the antiproliferative effects (combination index <1) of TMZ + SN‐38 (the active metabolite of CPT‐11) against colon cancer cells. Accordingly, GPI 15427 (40 mg/kg/day×5 days per os) in combination with TMZ (10 mg/kg/day×5 days) + CPT‐11 (4 mg/kg/day×5 days) significantly reduced the growth of tumor xenografts. Oral administration of GPI 15427 (40 mg/kg/q2 × 3 days) prevented intestinal injury and diarrhea induced by CPT‐11 (30 mg/kg/day × 3 days) reducing inflammation and PARP‐1 overactivation, as evidenced by immunohistochemical staining of intestinal tissue with antipoly(ADP‐ribose) antibody(Ab). Inconclusion, the PARP inhibitor represents a novel strategy to enhance the antitumor efficacy and reduce toxicity of chemotherapy in colon cancer.—Tentori, L., Leonetti, C., Scarsella, M., Muzi, A., Mazzon, E., Vergati, M., Forini, O., Lapidus, R., Xu, W., Dorio, A. S., Zhang, J., Cuzzo‐crea, S., Graziani, G. Inhibition of poly(ADP‐ribose) polymerase prevents irinotecan‐induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma. FASEB J. 20, E1024–E1036 (2006)

Prostate-specific membrane antigen (PSMA) enzyme activity is elevated in prostate cancer cells

Prostate‐specific membrane antigen (PSMA) is a glutamate carboxypeptidase that cleaves terminal carboxy glutamates from both the neuronal dipeptide N‐acetylaspartylglutamate (NAAG) and gamma‐linked folate polyglutamate. The prostate enzyme has activity in both the membrane and cytosolic fractions termed PSMA and PSMA′, respectively.

High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen

Surgical resection remains a definitive treatment for prostate cancer. Yet, prostate cancer surgery is performed without image guidance for tumor margin, extension beyond the capsule and lymph node positivity, and without verification of other occult metastases in the surgical field. Recently, several imaging systems have been described that exploit near-infrared (NIR) fluorescent light for sensitive, real-time detection of disease pathology intraoperatively. In this study, we describe a high-affinity (9 nM), single nucleophile-containing, small molecule specific for the active site of the enzyme PSMA. We demonstrate production of a tetra-sulfonated heptamethine indocyanine NIR fluorescent derivative of this molecule using a high-yield LC/MS purification strategy. Interestingly, NIR fluorophore conjugation improves affinity over 20-fold, and we provide mechanistic insight into this observation. We describe the preparative production of enzymatically active PSMA using a baculovirus expression system and an adenovirus that co-expresses PSMA and GFP. We demonstrate sensitive and specific in vitro imaging of endogenous and ectopically expressed PSMA in human cells and in vivo imaging of xenograft tumors. We also discuss chemical strategies for improving performance even further. Taken together, this study describes nearly complete preclinical development of an optically based small-molecule contrast agent for image-guided surgery.

Comparison of Neuropathy-Inducing Effects of Eribulin Mesylate, Paclitaxel, and Ixabepilone in Mice

Chemotherapy-induced neurotoxicity is a significant problem associated with successful treatment of many cancers. Tubulin is a well-established target of antineoplastic therapy; however, tubulin-targeting agents, such as paclitaxel and the newer epothilones, induce significant neurotoxicity. Eribulin mesylate, a novel microtubule-targeting analogue of the marine natural product halichondrin B, has recently shown antineoplastic activity, with relatively low incidence and severity of neuropathy, in metastatic breast cancer patients. The mechanism of chemotherapy-induced neuropathy is not well understood. One of the main underlying reasons is incomplete characterization of pathology of peripheral nerves from treated subjects, either from patients or preclinically from animals. The current study was conducted to directly compare, in mice, the neuropathy-inducing propensity of three drugs: paclitaxel, ixabepilone, and eribulin mesylate. Because these drugs have different potencies and pharmacokinetics, we compared them on the basis of a maximum tolerated dose (MTD). Effects of each drug on caudal and digital nerve conduction velocity, nerve amplitude, and sciatic nerve and dorsal root ganglion morphology at 0.25 × MTD, 0.5 × MTD, 0.75 × MTD, and MTD were compared. Paclitaxel and ixabepilone, at their respective MTDs, produced significant deficits in caudal nerve conduction velocity, caudal amplitude and digital nerve amplitudes, as well as moderate to severe degenerative pathologic changes in dorsal root ganglia and sciatic nerve. In contrast, eribulin mesylate produced no significant deleterious effects on any nerve conduction parameter measured and caused milder, less frequent effects on morphology. Overall, our findings indicate that eribulin mesylate induces less neuropathy in mice than paclitaxel or ixabepilone at equivalent MTD-based doses.

Head and neck cancer radiosensitization by the novel poly(ADP-ribose) polymerase inhibitor GPI-15427

In this study, we tested the ability of a novel poly(adenosine diphosphate ribose) polymerase (PARP) inhibitor, 10‐(4‐methyl‐piperazin‐1‐ylmethyl)‐2H‐7‐oxa‐1,2‐diaza‐benzo[de]‐anthracen‐3‐one (GPI‐15427), to enhance the effect of radiotherapy in a xenograft model of human head and neck squamous cell carcinoma (HNSCC).

Glutamate carboxypeptidase inhibition reduces the severity of chemotherapy-induced peripheral neurotoxicity in rat.

Chemotherapy is the most common method to treat cancer. The use of certain antineoplastic drugs, however, is associated with the development of peripheral neuropathy that can be dose-limiting. Excitotoxic glutamate release, leading to excessive glutamatergic neurotransmission and activation of N-methyl-d-aspartate (NMDA) receptors, is associated with neuronal damage and death in several nervous system disorders. N-Acetyl-aspartyl-glutamate (NAAG) is an abundant neuropeptide widely distributed in the central and peripheral nervous system which is physiologically hydrolyzed by the enzyme glutamate carboxypeptidase into N-Acetyl-aspartyl (NAA) and glutamate. Pharmacological inhibition of glutamate carboxypeptidase results in decreased glutamate and increased endogenous NAAG and has been shown to provide neuroprotection in several preclinical models. Here, we report the neuroprotective effect of an orally available glutamate carboxypeptidase inhibitor on three well-established animal models of chemotherapy (cisplatin, paclitaxel, bortezomib)-induced peripheral neuropathy. In all cases, glutamate carboxypeptidase inhibition significantly improved the chemotherapy-induced nerve conduction velocity deficits. In addition, morphological and morphometrical alterations induced by cisplatin and bortezomib in dorsal root ganglia (DRG) were improved by glutamate carboxypeptidase inhibition. Our data support a novel approach for the treatment of chemotherapy-induced peripheral neuropathy.

Inhibition by spermine of the inner membrane permeability transition of isolated rat heart mitochondria

The effect of spermine on the permeability transition of the inner mitochondrial membrane of isolated rat heart mitochondria was evaluated. The permeability transition was triggered using a series of agents (t‐butyl hydroperoxide, phenylarsine oxide, carboxyatractylate, and elevated Ca2+ and inorganic phosphate concentrations), and was monitored via Ca2+‐release, mitochondrial swelling and pyridine nucleotide oxidation. By all three criteria, spermine inhibited the transition. A C50 of 0.38 ± 0.06 (SD) mM was measured for inhibition.

Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression.

Progesterone receptor (PR) is an estrogen-stimulated gene which has a CpG island that is heavily methylated in a significant fraction of estrogen receptor (ER)-negative/PR-negative human breast cancers and cell lines, including MDA-MB-231 cells. Treatment of MDA-MB-231 cells with the demethylating agent, 5-aza-2′-deoxycytidine (deoxyC) led to demethylation and expression of ER and PR. However, simultaneous treatment with antiestrogen prevented PR transcription, suggesting that demethylation of PR alone is not sufficient to reactivate the PR gene. To examine the effects of ER on the methylation status of the PR CpG island, we stably transfected MDA-MB-231 cells with an inducible expression vector for ER. Surprisingly, in two cell clones, we found that induction of PR gene expression by ligand-bound ER does not require demethylation of the PR CpG island. In contrast, induction of PR transcription was inhibited by blocking the interaction of ER with SRC-1A, a coactivator of ER function. For the first time, we show that a transcription factor with the potential to remodel heterochromatin can activate gene expression without altering the methylation status of the CpG island. These results raise the possibility that demethylation and histone acetylation are distinct but complementary mechanisms for destabilizing heterochromatin and activating transcription.

Presence of isocitrate dehydrogenase mutations may predict clinical response to hypomethylating agents in patients with acute myeloid leukemia

Mutations in IDH1 and IDH2 occur in 15–20% of AML cases, resulting in the production of 2‐hydroxyglutarate, which promotes aberrant hypermethylation of DNA in leukemic cells. Although these mutations have been shown to have prognostic implications for patients with AML, optimal treatment strategies have yet to be defined. We retrospectively identified forty‐two patients with AML treated with DNA methyltransferase inhibitors (DNMTIs) decitabine (n = 36) or azacitidine (n = 6) and performed analysis of stored samples for the presence of IDH1 and IDH2 mutations. Of the forty‐two samples analyzed, seven (16.7%) had IDH mutations. Thirteen patients (31%) achieved remission [(complete remission (CR)/complete remission with incomplete count recovery (CRi)/partial response (PR)] after treatment with a DNMTI, five of seven (71.4%) with IDH mutations and eight of thirty‐five (22.9%) without IDH mutations (P = 0.01). When adjusted for age at diagnosis, sex, bone marrow blast percentage and cytogenetic, the odds of achieving response after administration of a DNMTI among patients with an IDH mutation was 14.2 when compared to patients without an IDH mutation (95%CI: 1.3–150.4). IDH1 and IDH2 mutations may predict a favorable response to DNMTI in patients with AML. Am. J. Hematol. 90:E77–E79, 2015.