Proof concept for clinical justification of network mapping for personalized cancer therapeutics

Abstract

To identify signature targets associated with patient-specific cancer lesions based on tumor versus normal tissue differential protein and mRNA coexpression patterns for the purpose of synthesizing cancer-specific customized RNA interference knockdown therapeutics. Analysis of biopsied tissue involved two-dimensional difference in-gel electrophoresis (2D-DIGE) analysis coupled with MALDI-TOF/TOF mass spectrometry for proteomic assessment. Standard microarray techniques were utilized for mRNA analysis. Priority was assigned to overexpressed protein targets with co-overexpressed genes with a high likelihood of functional nodal centrality in the cancer network as defined by the interactive databases BIND, HPRD and ResNet. HPLC-grade small interfering RNA (siRNA) duplexes were utilized to assess knockdown of target proteins in expressive cell lines as measured by western blot. Seven patients with metastatic cancer underwent biopsy. One patient (RW001) had biopsies from two disease sites 10 months apart. Seven priority proteins were identified, one for each patient (RACK 1, Ras related nuclear protein, heat-shock 27 kDa protein 1, superoxide dismutase, enolase1, stathmin1 and cofilin1). Prioritized proteins in RW001 from the two disease sites over time were the same. We demonstrated 480% siRNA inhibition of RACK 1 and stathmin1 of inexpressive malignant cell lines with correlated cell kill. Identification of functionally relevant target gene fingerprints, unique to an individual's cancer, is feasible 'at the bedside' and can be utilized to synthesize siRNA knockdown therapeutics. Further animal safety testing followed by clinical study is recommended.

Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy?

“…116 However, highly connected targets do allow for 'attack vulnerability.' In other words, the disordered circuitry characteristic of malignancy results in a change, such that the otherwise robust oncogenic process can become, almost paradoxically, more highly dependent on a specific rewired pathway ('pathway addiction').…”

“…In other words, the disordered circuitry characteristic of malignancy results in a change, such that the otherwise robust oncogenic process can become, almost paradoxically, more highly dependent on a specific rewired pathway ('pathway addiction'). 116 This thesis forms the theoretic basis of attempting to correct the tumor phenotype through the targeting of the dominant, offending oncogenic pathway.…”

Stathmin 1: a protein with many tasks. New biomarker and potential target in cancer

“…Based on differential proteogenomic signaling assessments of patients from our program [13,14], as well as published literature by others, there is a sound rationale for the therapeutic targeting of STMN1 in cancer patients [15]. Stathmin is highly expressed in a variety of human malignancies [16,17] and has been shown to be upregulated in multiple cancers.…”

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

“…The bifunctional construct bi-sh-STMN1 is directed against Stathmin1 (STMN1), a gene target candidate that is overexpressed in human cancer lines and was shown by us to be differentially overexpressed in cancer patients, based on mRNA and protein couplet signals in tumor/ normal tissue specimen analysis. 13 STMN1 is critically involved in mitotic spindle formation. 14,15 Previously, studies have shown that STMN1 knockdown by conventional siRNA resulted in G 2 /M cell cycle arrest, inhibition of clonogenicity and markedly increased apoptosis.…”