Rajat Bhattacharya

Endothelial Cells Promote the Colorectal Cancer Stem Cell Phenotype through a Soluble Form of Jagged-1

We report a paracrine effect whereby endothelial cells (ECs) promote the cancer stem cell (CSC) phenotype of human colorectal cancer (CRC) cells. We showed that, without direct cell-cell contact, ECs secrete factors that promoted the CSC phenotype in CRC cells via Notch activation. In human CRC specimens, CD133 and Notch intracellular domain-positive CRC cells colocalized in perivascular regions. An EC-derived, soluble form of Jagged-1, via ADAM17 proteolytic activity, led to Notch activation in CRC cells in a paracrine manner; these effects were blocked by immunodepletion of Jagged-1 in EC-conditioned medium or blockade of ADAM17 activity. Collectively, ECs play an active role in promoting Notch signaling and the CSC phenotype by secreting soluble Jagged-1.

Human mutations that confer paclitaxel resistance

The involvement of tubulin mutations as a cause of clinical drug resistance has been intensely debated in recent years. In the studies described here, we used transfection to test whether β1-tubulin mutations and polymorphisms found in cancer patients are able to confer resistance to drugs that target microtubules. Three of four mutations (A185T, A248V, R306C, but not G437S) that we tested caused paclitaxel resistance, as indicated by the following observations: (a) essentially 100% of cells selected in paclitaxel contained transfected mutant tubulin; (b) paclitaxel resistance could be turned off using tetracycline to turn off transgene expression; (c) paclitaxel resistance increased as mutant tubulin production increased. All the paclitaxel resistance mutations disrupted microtubule assembly, conferred increased sensitivity to microtubule-disruptive drugs, and produced defects in mitosis. The results are consistent with a mechanism in which tubulin mutations alter microtubule stability in a way that counteracts drug action. These studies show that human tumor cells can acquire spontaneous mutations in β1-tubulin that cause resistance to paclitaxel, and suggest that patients with some polymorphisms in β1-tubulin may require higher drug concentrations for effective therapy. Mol Cancer Ther; 9(2); 327–35

Therapeutic Silencing of KRAS Using Systemically Delivered siRNAs

Despite being among the most common oncogenes in human cancer, to date, there are no effective clinical options for inhibiting KRAS activity. We investigated whether systemically delivered KRAS siRNAs have therapeutic potential in KRAS-mutated cancer models. We identified KRAS siRNA sequences with notable potency in knocking down KRAS expression. Using lung and colon adenocarcinoma cell lines, we assessed antiproliferative effects of KRAS silencing in vitro. For in vivo experiments, we used a nanoliposomal delivery platform, DOPC, for systemic delivery of siRNAs. Various lung and colon cancer models were used to determine efficacy of systemic KRAS siRNA based on tumor growth, development of metastasis, and downstream signaling. KRAS siRNA sequences induced >90% knockdown of KRAS expression, significantly reducing viability in mutant cell lines. In the lung cancer model, KRAS siRNA treatment demonstrated significant reductions in primary tumor growth and distant metastatic disease, while the addition of CDDP was not additive. Significant reductions in Ki-67 indices were seen in all treatment groups, whereas significant increases in caspase-3 activity were only seen in the CDDP treatment groups. In the colon cancer model, KRAS siRNA reduced tumor KRAS and pERK expression. KRAS siRNAs significantly reduced HCP1 subcutaneous tumor growth, as well as outgrowth of liver metastases. Our studies demonstrate a proof-of-concept approach to therapeutic KRAS targeting using nanoparticle delivery of siRNA. This study highlights the potential translational impact of therapeutic RNA interference, which may have broad applications in oncology, especially for traditional “undruggable” targets. Mol Cancer Ther; 13(12); 2876–85. ©2014 AACR.

The requirement for freshly isolated human colorectal cancer (CRC) cells in isolating CRC stem cells

Background:Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs.Methods:Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment.Results:None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not.Conclusions:PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.

Molecular Basis for Class V β-Tubulin Effects on Microtubule Assembly and Paclitaxel Resistance

Vertebrates produce at least seven distinct β-tubulin isotypes that coassemble into all cellular microtubules. The functional differences among these tubulin isoforms are largely unknown, but recent studies indicate that tubulin composition can affect microtubule properties and cellular microtubule-dependent behavior. One of the isotypes whose incorporation causes the largest change in microtubule assembly is β5-tubulin. Overexpression of this isotype can almost completely destroy the microtubule network, yet it appears to be required in smaller amounts for normal mitotic progression. Moderate levels of overexpression can also confer paclitaxel resistance. Experiments using chimeric constructs and site-directed mutagenesis now indicate that the hypervariable C-terminal region of β5 plays no role in these phenotypes. Instead, we demonstrate that two residues found in β5 (Ser-239 and Ser-365) are each sufficient to inhibit microtubule assembly and confer paclitaxel resistance when introduced into β1-tubulin; yet the single mutation of residue Ser-239 in β5 eliminates its ability to confer these phenotypes. Despite the high degree of conservation among β-tubulin isotypes, mutations affecting residue 365 demonstrate that amino acid substitutions can be context sensitive; i.e. an amino acid change in one isotype will not necessarily produce the same phenotype when introduced into a different isotype. Modeling studies indicate that residue Cys-239 of β1-tubulin is close to a highly conserved Cys-354 residue suggesting the possibility that disulfide formation could play a significant role in the stability of microtubules formed with β1- but not with β5-tubulin.

Mitotic Centromere-associated Kinesin (MCAK) Mediates Paclitaxel Resistance

Background: Mutations causing paclitaxel resistance stimulate microtubule detachment from centrosomes. Results: Depletion of mitotic centromere-associated kinesin (MCAK) reverses microtubule detachment and paclitaxel resistance. Conclusion: MCAK plays a pivotal role in the mechanism of microtubule detachment and drug resistance. Significance: The ability of MCAK to reverse paclitaxel resistance identifies modulators of microtubule detachment as important new drug targets. Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III β-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.

A minor β-tubulin essential for mammalian cell proliferation

Mammals use tubulin from multiple genes to construct microtubules. Some genes are expressed in a tissue specific manner, while others are expressed in almost all cell types. beta5-Tubulin is a minor, ubiquitous isoform whose overexpression was recently shown to disrupt microtubules. Using inhibitory RNA, we now report that suppression of beta5 production in both human and hamster cells blocks cell proliferation. Cells depleted of beta5 either trigger the mitotic checkpoint and undergo apoptosis; or they experience a transient mitotic block, a high incidence of lagging chromosomes, and progression into G1 without cytokinesis to become large, flat cells with elevated DNA content. Microtubules appear to be normally organized in cells depleted of beta5, but they are rich in acetylated alpha-tubulin indicating that they may be more stable than normal. The results provide the first evidence that a specific isoform of beta-tubulin is required for mitosis.

ATP Citrate Lyase Mediates Resistance of Colorectal Cancer Cells to SN38

Combination chemotherapy is standard for metastatic colorectal cancer; however, nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may enable identification of novel targets and more effective therapy. Irinotecan is commonly used in first- and second-line therapy for patients with metastatic colorectal cancer, with the active metabolite being SN38. Emerging evidence suggests that altered metabolism in cancer cells is fundamentally involved in the development of drug resistance. Using Oncomine and unbiased proteomic profiling, we found that ATP citrate lyase (ACLy), the first-step rate-limiting enzyme for de novo lipogenesis, was upregulated in colorectal cancer compared with its levels in normal mucosa and in chemoresistant colorectal cancer cells compared with isogenic chemo-naïve colorectal cancer cells. Overexpression of exogenous ACLy by lentivirus transduction in chemo-naïve colorectal cancer cells led to significant chemoresistance to SN38 but not to 5-fluorouracil or oxaliplatin. Knockdown of ACLy by siRNA or inhibition of its activity by a small-molecule inhibitor sensitized chemo-naïve colorectal cancer cells to SN38. Furthermore, ACLy was significantly increased in cancer cells that had acquired resistance to SN38. In contrast to chemo-naïve cells, targeting ACLy alone was not effective in resensitizing resistant cells to SN38, due to a compensatory activation of the AKT pathway triggered by ACLy suppression. Combined inhibition of AKT signaling and ACLy successfully resensitized SN38-resistant cells to SN38. We conclude that targeting ACLy may improve the therapeutic effects of irinotecan and that simultaneous targeting of ACLy and AKT may be warranted to overcome SN38 resistance. Mol Cancer Ther; 12(12); 2782–91. ©2013 AACR.

Class V β-tubulin alters dynamic instability and stimulates microtubule detachment from centrosomes

The need for multiple tubulin genes in vertebrate organisms is poorly understood. This article shows that a minor, ubiquitious β-tubulin isotype strongly influences microtubule plasticity by altering dynamic behavior and the stability of microtubule attachment to centrosomes.

Cell cycle dependent degradation of MCAK: evidence against a role in anaphase chromosome movement.

MCAK, a kinesin related motor protein with microtubule depolymerizing activity, is known to play an important role in spindle assembly and correcting errors in mitotic chromosome alignment. Experiments to determine how cellular levels of the protein are regulated demonstrate that MCAK accumulates during cell cycle progression, reaches a maximum at G2/M phase, and is rapidly degraded by the proteasome during mitosis. Immunofluorescence microscopy further indicates that MCAK largely disappears from kinetochores and spindle poles at the metaphase to anaphase transition. A phosphorylated form of MCAK appears during mitosis and seems to be preferentially degraded, but degradation does not appear to depend on Aurora B, a kinase reported to be involved in regulating the error correcting activity of the protein. These studies indicate that MCAK activity is limited during the latter stages of mitosis by protein degradation, and argue against a role for the protein in anaphase chromosome movement.