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LITERATURE OVERVIEW

This educational resource provides a detailed overview of key scientific studies highlighting some of the unique properties of CDK4 & 6.

CDK4, CDK6 & Cell Proliferation

Similarities and Differences in Normal Cells and Cancer Cells

CDK4 and CDK6 are activated by various mitogenic signaling pathways to regulate cell-cycle progression via interactions with the Rb-E2F pathway.1,2 The relative importance of CDK4 and CDK6 varies in different types of normal and cancerous tissues.3-9 Preclinical studies suggest that CDK4 plays a pivotal role in breast cancer.6,7 Here we review key articles highlighting the differences between CDK4 and CDK6 in order to elucidate potential implications that could improve our understanding of breast cancer in relation to CDK4 & 6.

Cyclin-dependent kinases

Key regulatory enzymes of the cell cycle and gene transcription

The cell cycle drives mitosis and cell proliferation. Normally, cells are maintained in the quiescent state, G0, unless highly regulated physiological cues trigger cell-cycle reentry.1 Cyclin-dependent kinases (CDKs) are key regulatory enzymes that drive cell-cycle reentry and progression.1 CDKs are activated by small proteins called cyclins.1

To date, approximately 20 different CDKs have been identified, each with unique regulatory roles in the cell cycle and gene transcription. The roles of different CDKs in the cell cycle are explained below.1

Cell-cycle reentry is controlled by CDK4 and CDK6, which trigger transition from the G0/G1 phase to the S phase of the cell cycle. CDK4 and CDK6 can be activated by a number of mitogenic signaling pathways that promote expression of D-type cyclins and stabilization of the CDK-cyclin complex.1

This complex inactivates the retinoblastoma tumor suppressor protein (Rb), releasing the E2F transcription factor, initiating the transcription of genes that facilitate cell-cycle progression.1

Deregulation of the CDK4 & CDK6-Rb pathway is a key driver in many cancers

The CDK4 & CDK6-Rb pathway has been implicated in approximately 30 different types of cancers, including the 3 most common: breast, colon, and lung cancer.1,2

Deregulation of the CDK4 & CDK6-Rb axis may occur by a variety of mechanisms including1:

  • Loss of Rb protein expression leading to uncontrolled E2F gene transcription
  • Upregulation of CDK4 and cyclin D1
  • Loss of proteins that inhibit the activities of CDK4 and/or CDK6

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CDK4 and CDK6

Different roles in normal cells

Historically, CDK4 and CDK6 were thought to be functionally equivalent.1,2

However, recent studies have found that CDK4 and CDK6 actually have different expression profiles in different tissue types and these differences are seen in both normal and cancerous tissues.3-9 A better understanding of these differences and their implications in specific malignancies may inform future therapeutic developments that could one day benefit people with cancer.

CDK4: important in the development of pancreatic and pituitary cells

Preclinical studies have found key differences in the roles and functions of CDK4 and CDK6 in specific non-malignant cell types. For example, in mouse models, loss of CDK4 selectively impaired proliferation of specific cell types in the pancreas and anterior pituitary gland (Fig. 1).3,4

Pancreatic beta-islet cell mass

Pituitary cell mass by region

Figure 1. (A) In mouse models, CDK4 knockout (CDKn/n) inhibited postnatal proliferation of pancreatic beta-islet cells at 4 months compared to wild type (CDK4+/+), and restoration of a highly active form of CDK4 (CDK4R/R) restored proliferation.3 (B) CDK4 knockout (CDKn/n) inhibited proliferation of cells in the adenohypophysis of the pituitary gland and this was reversed by restoration of CDK4 expression (CDK4R/R).3

In this study, mouse pancreatic islet cells expressed very low levels of CDK6.3 This was shown to be insufficient to drive proliferation in the absence of CDK4.3 In the anterior pituitary cells, knockout of CDK4 inhibited proliferation despite normal levels of CDK6.3 In another study of the anterior pituitary, CDK6 knockout in mice did not affect proliferation when CDK4 expression was maintained.4 These studies provide evidence that CDK4 and CDK6 are not functionally redundant in certain cell types.

CDK6: important in hematologic cells

CDK6 appears to play a unique role in the differentiation and proliferation of hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). In a study by Scheicher et al, CDK6 expression was important for differentiation and proliferation of HSCs and LSCs.5

When CDK6+/+ and CDK6-/- HSCs were transplanted at varying ratios into mice whose bone marrow was lethally irradiated, the majority of new leukocytes originated from CDK6+/+ HSCs regardless of the ratio of CDK6+/+ to CDK6-/- cells transplanted. The presence of CDK4 in these irradiated mice did not compensate for the loss of CDK6 (Fig. 2).5

Figure 2. When CDK6-/- and CDK6+/+ HSCs were transplanted at varying ratios into lethally irradiated mice, the majority of new leukocyte cells were derived from the CDK6+/+ cells even when the majority of transfected HSCs were CDK6-/-.5

In this same study, transplantation of BCR/ABL-infected bone marrow from CDK6-/- mice failed to induce a leukemic phenotype, despite the fact that the recipient mice did harbor leukemic stem cells.5 Based on these results, the investigators concluded that CDK6 is essential for the proliferation of HSCs and LSCs and that the roles of CDK4 and CDK6 are clearly nonredundant in these cell types.5

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CDK4 & CDK6: key differences in breast cancer

Multiple studies have demonstrated the importance of CDK4 in breast cancer.

In a study by Yu et al, Western blot analysis revealed that CDK4 expression was higher in breast tumors from mice compared to normal mouse mammary tissue; the levels of CDK6 expression in breast tumors were lower than that of CDK4.6

In breast cancer xenograft models, breast tumor formation and growth was essentially inhibited in mice inoculated with CDK4-deficient tumor cells, despite normal levels of CDK6 (Fig. 3).6 CDK6-deficiency did not have a similar effect on breast tumor formation in the presence of normal CDK4 expression.

Figure 3. Breast tumor formation and tumor weight were decreased in CDK4-/CDK6+ (anti-CDK4) mice compared with CDK4+/CDK6+ (Contr) mouse models, indicating the essential role of CDK4 in breast cancer.6

While Yu et al demonstrated the importance of CDK4 in breast tumor formation and growth, another study by Lucas et al found that normal human breast cells expressed high levels of CDK6, whereas cancerous breast cells had reduced CDK6 expression, with several cancer cell lines expressing almost no CDK6.7 Quantitative analysis of these samples revealed that CDK6 levels and kinase activity were significantly lower in all cancer cell lines relative to the normal breast cell line.

Furthermore, Lucas et al found that altering certain breast cancer cell lines to express normal levels of CDK6 actually reduced cell proliferation compared with the unaltered parent cell lines that expressed low levels of CDK6 (Fig. 4).7

453 parent line

468 parent line

Figure 4. Breast cancer cell lines expressing normal levels of CDK6 ("wt" lines) had decreased proliferation compared with those unaltered cell lines expressing low levels of CDK6 ("453" parent line, "468" parent line, and "dn" lines).7

In addition to having different effects on breast cancer cell proliferation, CDK4 & CDK6 were found to have different effects on steroid hormone production in a breast cancer cell line.8 Overexpression of CDK4 resulted in upregulation of steroid metabolic enzymes creating a “proestrogenic” state. This may create a favorable growth environment for estrogen-dependent breast cancer cells. The opposite effect was seen when CDK6 was overexpressed.8

In another study by Grigoriadis et al, CDK4 expression was elevated in samples of human breast tumors relative to CDK6. In normal breast epithelium, the opposite expression profile was observed.9 These data are shown in the chart below (Fig. 5).

Gene Normal Luminal Epithelium Malignant Breast Epithelium P Value
CDK4 30 tpm 64 tpm 0.001
CDK6 40 tpm 17 tpm 0.003

tpm=transcripts per million mapped reads.

Figure 5. Analysis of gene expression in normal human breast tissue vs breast tumor tissue samples confirmed significantly different levels of CDK4 & CDK6 gene transcripts in cancerous vs normal breast tissue.9

CDK4, CDK6 & Cell Proliferation

Similarities and Differences in Normal Cells and Cancer Cells

Content Summary

CDK4 & CDK6 are activated by a number of different mitogenic signaling pathways and regulate cell-cycle progression via interaction with the Rb-E2F pathway1,2

The relative importance of CDK4 and CDK6 differs in different types of normal and cancerous cells3-7,9

Preclinical studies suggest CDK4 plays a pivotal role in proliferation and steroid hormone production in breast cancer8

These findings suggest there are key differences between CDK4 and CDK6 that may have important implications for our understanding of breast cancer

The answer is True.
Activated CDK4 & CDK6 phosphorylate Rb releasing it from E2F transcription factor. This leads to transcription of genes necessary for cell replication including regulatory cyclins and CDKs as well as other regulatory proteins.1

References: 1. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. Nat Rev Drug Discov. 2015;14(2):130-146. 2. Ortega S, Malumbres M, Barbacid M. Biochimica et Biophysica Acta. 2002;1602:73-87. 3. Martín J, Hunt SL, Dubus P, et al. Oncogene. 2003;22:5261-5269. 4. Jirawatnotai S, Aziyu A, Osmundson EC, et al. J Biol Chem. 2004;279:51100-51106. 5. Scheicher R, Hoelbl-Kovacic A, Bellutti F, et al. Blood. 2015;125:90-101. 6. Yu Q, Sicinska E, Geng Y, et al. Cancer Cell. 2006;9:23-32. 7. Lucas JJ, Domenico J, Gelfand EW. Mol Cancer Res. 2004;2:105-114. 8. Jia Y, Domenico J, Swasey C, Wang M, Gelfand EW, Lucas JJ. PLoS ONE. 2014;9:e97448. 9. Grigoriadis A, Mackay A, Reis-Filho JS, et al. Breast Cancer Res. 2006;8(5):R56.

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